CN104402988A - Recombinant variant and preparation method of human interleukin-29 - Google Patents
Recombinant variant and preparation method of human interleukin-29 Download PDFInfo
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- CN104402988A CN104402988A CN201410756203.3A CN201410756203A CN104402988A CN 104402988 A CN104402988 A CN 104402988A CN 201410756203 A CN201410756203 A CN 201410756203A CN 104402988 A CN104402988 A CN 104402988A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
Abstract
The invention provides a recombinant hIL-29 variant (rhIL-29L) of human interleukin-29 (hIL-29) of which the 33th-site lysine of mature peptide is subjected to site-directed mutagenesis to form arginine and a preparation method of the recombinant hIL-29 variant, and belongs to the field of bioengineering. The specific preparation method provided by the invention comprises the following steps: 1) manually designing and synthesizing an upstream primer, a downstream primer and a site-directed mutagenesis primer of the mature peptide of the hIL-29; 2) performing site-directed mutagenesis on the 98th-site base of an encoding gene of the mature peptide of the hIL-29 by using the upstream primer, the downstream primer and the site-directed mutagenesis primer; 3) constructing a recombinant eukaryon expression vector which contains the encoding gene of the hIL-29 variant, and converting pichia pastoris to obtain recombinant yeast engineered bacteria; 4) culturing the recombinant yeast engineered bacteria, and adding 1.5 percent (v/v) methanol into culture liquid to induce the recombinant yeast engineered bacteria to express the hIL-29 variant; 5) purifying the hIL-29 variant from the supernatant of the culture liquid by adopting SP-Sepharose Fast Flow cation-exchange chromatography and Sephadex G-75 gel filtration chromatography. The recombinant yeast engineered bacteria constructed by the preparation method can be used for high-density culture and secretory expression of rhIL-29, and is suitable for industrial preparation of the variant of the mature peptide of the rhIL-29.
Description
Technical field
The invention provides a kind of Human Inter Leukin-2 9 mature peptide the 33rd Methionin rite-directed mutagenesis is arginic restructuring varient (rhIL-29L) and preparation method thereof, belongs to bioengineering field.
Background technology
Human Inter Leukin-2 9 (interleukin 29, IL-29) is a kind of new cytokine in recent years found, its structure and function and I type Interferon, rabbit (interferon, IFN) similar, be therefore also called IFN-λ 1.
The zcyto21 assignment of genes gene mapping is in No. 19 chromosome long arm (19q13.13), comprise 5 exons, its gene structure is similar to the gene structure of IL-10 family member, but has notable difference with the gene structure (only containing an exon) of I type Interferon, rabbit.IL-29 forms by containing 19 amino acid whose signal peptides and 181 amino acid whose mature peptides, and its mRNA total length 856bp, opening code-reading frame is 603bp.The relative molecular mass of IL-29 is about 20000 ~ 33000, has the N-connection glycosylation site that potential, has two disulfide linkage.Disulfide linkage is to the correct folding of IL-29 and biologic activity is very important.
Zcyto21 is combined by the receptor complex on cytolemma, the transduction of start signal and performance biological action.IL-29 and IL-28 shares a heterodimeric receptor mixture be made up of IL-28R1 and IL-10R2, and wherein IL-28R1 is binding subunit, and IL-10R2 is auxiliary subunit.IL-28R1 is specific binding subunit in the shared acceptor of IL-29 and IL-28, to the cell response of IL-29, there is specificity, auxiliary subunit IL-10R2 is the acceptor integral part of I type Interferon, rabbit, IL-10, IL-22 and IL-26, it generally expresses in hematopoiesis and non-hematopoietic cell, to the cell response of IL-29 without specificity.
Identical Jak-STAT (janus kinase-signal transducer of transcription) signal transduction path shared by zcyto21 and I type Interferon, rabbit, activate identical Jak-STAT signal transduction pathway, promote the genetic expression that one group common.First IL-29 forms IL28R1-IL29-IL10R2 ternary complex with its receptors bind, then by being positioned at intracellular two receptor chain transduction signals, initiating signal cascade reaction, make the tyrosine residues phosphorylation of STAT1, STAT2, STAT3, STAT4 and STAT5, ISGF3 (IFN-stimulated gene factor 3complex) is impelled to enter nucleus and ISRE (IFN-stimulatedresponse element) interacts, thus the transcribing of regulatory gene.Therefore, IL-29 shows some character identical with I type Interferon, rabbit, as biologic activity such as antiviral, antiproliferative, anti-tumor in vivo and immunomodulatorys.
Research finds, zcyto21 is mainly expressed in epithelial cell, in human blood, brain, lung, pancreas and testis, then have low-level expression.The hematopoiesis of Different Origin and non-hematopoietic cell are by after various virus infection, can abduction delivering IL-29, having found that there is Measles virus, mumps virus, myocarditis virus, influenza A virus, Sendai virus, new castle disease virus, strand (+) RNA viruses, double-stranded DNA virus etc. at present can inductive infection cell expressing IL-29.
IFN be find the earliest, most study, first cloning and the cytokine for clinical treating disease.At present, IFN is mainly used in the treatment of some tumour and virus disease, but very large to the Different therapeutical effect of different tumours and virus disease, and often occurs untoward reaction.The structure and function of IL-29 is similar to I type IFN, and can optionally act on dissimilar target cell, it can be used as the treatment that the substitute of I type IFN (IFN-α, IFN-β) or supplement are used for some disease, in tumour, organ transplantation, autoimmune disorder and allergic disorder etc. are prevented and treated, have potential using value.
Summary of the invention
The invention provides a kind of hIFN-LAMBDA1 mature peptide the 33rd Methionin rite-directed mutagenesis is arginic varient (hIL-29L) and preparation method thereof.
Technical scheme provided by the invention is as follows:
(1) a first aspect of the present invention, a kind of hIL-29 of inserting mature peptide the 33rd Methionin rite-directed mutagenesis is provided to be the recombinant eukaryon expression vector of arginic varient encoding gene, by hIL-29 varient encoding gene (SEQ IDNo:2) of the present invention and pPIC9K
m(come from Invitrogen company, through this laboratory transformation also patent applied for, application number is: 201110410391.0) recombinate, and obtains recombinant eukaryon expression vector pPIC9K
m-hIL-29L, as shown in Figure 1; This recombinant vectors expression product is that in peptide chain, the 33rd Methionin rite-directed mutagenesis is arginic hIL-29 varient (SEQ ID No:1); And the present invention is by pPIC9K
malpha factor in the recognition site GAG AAA AGA of restriction enzyme Xho I recognition site CTC GAG and Proteinase K ex2, introduced the aminoterminal of hIL-29 varient encoding gene (SEQ ID No:2) by PCR method, the peptide chain of the hIL-29 varient that this recombinant eukaryon expression vector is expressed (SEQ ID No:1) can not increase extra amino-acid residue because introducing restriction endonuclease sites.
(2) a second aspect of the present invention, there is provided a kind of recombination yeast engineering bacteria GS115/hIL-29L expressing zcyto21 varient, this project bacterium is Pichia pastoris GS115 (Invitrogen company) recombinant eukaryon expression vector pPIC9K described by the present invention
m-hIL-29L transforms and obtains, and pPIC9K
min signal peptide gene sequence and the encoding gene of hIL-29 varient be all incorporated in the genome of this project bacterium, therefore this project bacterium can secretion expression hIL-29 varient in nutrient solution.
(3) a third aspect of the present invention, provide a kind of method preparing hIL-29 varient mature peptide, the method comprises the following steps:
1. cultivate above-mentioned recombination yeast engineering bacteria, in nutrient solution, add the methyl alcohol of 1.5% (v/v) as inductor, induction recombination yeast engineering bacteria secreting, expressing hIL-29 varient;
2. centrifugation medium removes thalline and insolubles, collects nutrient solution supernatant and removes Ficus caricaL through ultrafiltration and concentration and dialysis;
3. the hIL-29 varient of the culture supernatant after desalination in strong cation exchange chromatography column and gel chromatography column isolation and purification culture liquid, collects the elutriant containing hIL-29 varient;
4. the hIL-29 varient of SDS-PAGE and Western Blot methods analyst purification Identification is used.
Accompanying drawing explanation
Fig. 1 is eukaryotic expression recombinant plasmid pPIC9K
mthe structure collection of illustrative plates of-hIL-29L
Fig. 2 is the pcr amplification result of IL-29 varient encoding gene
Fig. 3 is the SDS-PAGE detected result of restructuring hIL-29 varient
Fig. 4 is the Western Blot detected result of restructuring hIL-29 varient
Embodiment
Below in conjunction with specific embodiment, further set forth working method of the present invention, these embodiments only for describing the present invention in detail, and are not used in and limit the scope of the invention.
In following embodiment, the synthesis of all PCR primer and the mensuration of DNA sequence dna complete by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; Substratum used is all prepared by the formula of Pichia anomala expression handbook (Invitrogen company) if no special instructions.
The determination in embodiment one mutational site
1, the general introduction of energy is freely combined
Freely combining of protein-protein complex can be subject to the impact of multiple different factor, comprises the electrostatic interaction between albumen and receptor protein and hydrophobic interaction.Avidity is determined by net effect, is balanced and optimize each energy term to obtain strong bonding force by protein engineering.At present, the mutant obtaining high-affinity by modifying protein becomes one of study hotspot.
The present invention adopts Bo Sen-Boltzmann (Poisson-Boltzmann, PB) continuous model is carried out electrostatic calculations and is analyzed by COMBINE (Comparative Binding Energy), acquisition system specificity quantitative structure-activity relation model (QSAR, quantitative structure-activity relationship), this model can be used for estimating that overall freely combination can difference (electrostatic force and non-electrostatic force).The basic thought of QSAR method sets up a regression model based on the biological activity of part physicochemical property to a large amount of related complex, thus predict the properties of given molecule.PB is that molecular mechanics analyzes the method for relative free energy between Conjugated free energy with different conformation in conjunction with continuum solvent model in recent years, with the total energy of solute system in molecular mechanics and dynamics vacuum, with the electrostatic term Δ G of Poisson-Boltzmann (PB) equation solution solvation number
ele binding, Δ G
ele binding=Δ G
ele desol-bn+ Δ G
ele desol-bs+ E
ele bn-bs, ask nonpolar moiety item Δ Gnp in solvation number according to the empirical relationship between the palp surface-area of solvent and hydrophobic free energy, everyly add system's entropy effect-T Δ S above, Δ G can be expressed as
bind=Δ E
mM+ Δ G
slov-T Δ S, Δ G
slov=Δ G
ele binding+ Δ Gnp.In COMBINE analyzes, the interaction energy between acceptor and part is decomposed on the basis of base pair.The ANAL modulus of AMBER7.0 is used to calculate the Lennard-Jones interaction energy between Coulomb's force and each energy minimization protein residues.
2, the calculating of energy is freely combined
Solvation energy formula is defined as follows: Δ Δ G
sol=Δ G
sol-com-Δ G
sol1-Δ G
sol2
Coulomb's force formula is defined as follows: Δ Δ G
coul=Δ G
coul-com-Δ G
coul1-Δ G
coul2
Conjugated free energy is defined as follows: Δ Δ G
bind=Δ Δ G
sol+ Δ Δ G
coul
Wherein: Δ G
sol-comrepresent the solvation energy of mixture, Δ G
sol1represent the solvation energy of part, Δ G
sol2represent the solvation energy of acceptor; Δ G
coul-comrepresent the solvation energy of mixture, Δ G
coul1represent the solvation energy of part, Δ G
coul2represent the solvation energy of acceptor.The Conjugated free energy that the present invention records mature peptide IL-29 by APBS is-1220kJ/mol; The solvation energy of the IL-29 varient part that the 33rd amino acids is undergone mutation is-6.44E+02kJ/mol, and Coulomb's force is-6.70E+02, and freely combining can be-1310kJ/mol.
3, mutational site
By to the analysis freely combining energy in the present invention, determine that human interleukin-2 9 the 33rd amino acids is mutational site.
The clone of embodiment two zcyto21 varient encoding gene
1, design and synthetic pcr primer thing
According to the zcyto21 gene order in GenBank and pPIC9K
mthe alpha factor signal peptide sequence of carrier, with Oligo 7 software design a pair Auele Specific Primer and mutant primer as follows:
Upstream primer (hIL-29-F): 5 '-
cTCGAGAAAAGAgGCCCTGTCCCCACTTCC-3 '
Downstream primer (hIL-29-R): 5 '-
gCGGCCGCtCAGGTGGACTCAGGGTGG-3 '
Mutant primer (hIL-29-A): 5 '-CGTCCCTGG
ccCTCTTGAAGCTCG-3 '
The recognition site (GAGAAAAGA) of restriction enzyme Xho I recognition site (CTCGAG) and Proteinase K ex2 is added in upstream primer, add Not I site (GCGGCCGC) in downstream primer, the size of amplified production is about 560bp.
2, the clone of zcyto21 varient encoding gene
Adopt large primer PCR method, rite-directed mutagenesis is carried out to hIL-29 mature peptide gene.The first round, pcr amplification was with pPIC9K
m-hIL-29 plasmid is template, hIL-29-F and hIL-29-A is primer, and reaction conditions is as follows: 94 DEG C of denaturation 2min; 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 1min, 30 circulations; 72 DEG C extend 10min.Second takes turns large primer PCR amplification with pPIC9K
m-hIL-29 plasmid is template, and with first round PCR primer and hIL-29-R for primer, reaction conditions is identical with the first round.Amplified production is through 1% agarose gel electrophoresis analysis, and result as shown in Figure 2; The goal gene fragment that purifying is about 560bp is reclaimed with EZ-10SpinColumn DNA Extraction Kit (TaKaRa company); The goal gene fragment reclaimed is connected with cloning vector pUCm-T (BBI company), construction recombination plasmid pUCm-T-hIL-29L transform competent E. coli JM109, blue hickie screening positive transformant, serve the order-checking of extra large Sangon company, sequencing result is as shown in SEQ IDNo:2.Sequencing result is through DNAMAN software analysis and compare with the cDNA sequence of the zcyto21 in GenBank, goal gene in result display recombinant plasmid pUCm-T-hIL-29L has 1 base to morph, this base morphed is: the 98th bit base sports G (guanine) by A (VITAMIN B4), AGG is sported by AAG by the triplet code of this based composition, 33rd amino acids residue of this triplet code coding sports arginine (Arg) by Methionin (Lys), as shown in SEQ ID No:1.
The recombinant eukaryon expression vector of embodiment three construction expression zcyto21 varient
Get plasmid pUCm-T-hIL-29L and carrier pPIC9K
m(come from Invitrogen company, through this laboratory transformation also patent applied for, application number is: 201110410391.0).Carry out double digestion with restriction enzyme Xho I and Not I respectively, reclaim goal gene fragment and the carrier pPIC9K of about 560bp
mlarge fragment, use T
4dNA ligase (TaKaRa company) connects 16h in 16 DEG C of water-baths, will connect product conversion competence e. coli jm109, and obtain recombinant expression plasmid pPIC9K through PCR Screening and Identification
m-hIL-29L, serve the order-checking qualification of extra large Sangon company, sequencing result confirms pPIC9K
mcompletely the same in the coding gene sequence of the hIL-29 varient in-hIL-29L and pUCm-T-hIL-29L.
The screening of embodiment four eukaryotic expression recombinant plasmid transformed yeast GS115 and high copy recombinant bacterial strain thereof
Extract the pPIC9K through order-checking qualification
m-hIL-29L, after cutting with restriction enzyme Sal I enzyme, the agarose gel electrophoresis with 1% is separated and reclaims linearizing pPIC9K
m-hIL-29L; By linearizing pPIC9K
m-hIL-29L mixes with yeast GS115 competent cell, carries out electricity transform according to the method for Pichia anomala expression handbook (Invitrogen company).
Converted product is coated not containing the MD culture plate of His, cultivate 2-3 days for 30 DEG C, select the YPD that the vigorous dominant colony of MD grow on plates is inoculated in containing 2mg/mL G418 dull and stereotyped, cultivate 2-3 days for 30 DEG C, select the YPD that eugonic dominant colony is inoculated in containing 4mg/mL G418 dull and stereotyped, cultivate 2-3 days for 30 DEG C, obtain the pichia spp recombinant bacterial strain GS115/hIL-29L of high copy integration from the YPD plate screening containing 4mg/mL G418.
The abduction delivering of embodiment five zcyto21 varient, purifying and analysis
Recombinant bacterial strain GS115/hIL-29L is inoculated BMGY substratum, in 30 DEG C, shaking culture 24h under 220rpm/min condition, by centrifugal for nutrient solution room temperature 3000g 5min collecting cell, with 25mL BMMY re-suspended cell, in 30 DEG C, 220rpm/min continues cultivation; After Transition medium, the methyl alcohol adding 1.5% every 24h in nutrient solution carries out abduction delivering, cultured continuously 60h.Collected by centrifugation culture supernatant, first detect the expression of hIL-29 varient in culture supernatant with SDS-PAGE, then use goat-anti zcyto21 polyclonal antibody (R & D company) to carry out Western Blot and identify the hIL-29 varient of expressing.By culture supernatant ultrafiltration and concentration (molecular weight that dams is 10kDa), use SP-Sepharose Fast Flow ion exchange chromatography and Sephadex G-75 gel filtration chromatography again, purified product detects through SDS-PAGE and Western Blot identifies, the restructuring hIL-29 varient of SDS-PAGE result display purifying is single protein band, its molecular weight is 22.6kDa, as shown in Figure 3; Western blotting result display single reaction band, as shown in Figure 4.
Claims (6)
1. recombinant human interleukin--2 9 varient (rhIL-29L) in the present invention is characterised in that in the IL-29 peptide chain of expression, the 33rd amino acids Methionin (Lys) is arginine (Arg) through artificial rite-directed mutagenesis, corresponding to the SEQ ID NO:1 in sequence table.
2. in the present invention through the coding gene sequence of manually Fixedpoint mutation modified Human Inter Leukin-2 9 varient, it is characterized in that in this sequence, the 98th bit base sports G (guanine) by A (VITAMIN B4), AGG is sported by AAG, corresponding to the SEQ ID NO:2 in sequence table by the triplet code of this based composition.
3. express the recombinant eukaryon expression vector of recombinant human interleukin--2 9 varient in the present invention, it is characterized in that the encoding gene inserting Human Inter Leukin-2 9 varient according to claim 2 in described recombinant eukaryon expression vector.
4. the recombinant yeast pichia pastoris engineering bacteria in the present invention, it is characterized in that the encoding gene being integrated with Human Inter Leukin-2 9 varient according to claim 2 in the karyomit(e) of described recombinant yeast pichia pastoris, and excreting and expressing recombinant human interleukin II 9 varient is in nutrient solution.
5. prepare a method for recombinant human interleukin--2 9 varient, it is characterized in that, comprise step:
(1) with suitable nutrient solution, recombinant yeast pichia pastoris engineering bacteria according to claim 4 is cultivated;
(2) in nutrient solution, suitable inductor is added, induction recombinant yeast pichia pastoris engineering bacteria excreting and expressing recombinant human INF-LUMBDA1 varient;
(3) from nutrient solution, separation and purification goes out recombinant human interleukin--2 9 varient of recombinant yeast pichia pastoris secreting, expressing.
6. method as claimed in claim 5, is characterized in that, step (1) nutrient solution used is BMGY nutrient solution and BMMY nutrient solution; Step (2) inductor used is methyl alcohol, and the final concentration adding methyl alcohol is 1.5% of nutrient solution volume; Step (3) separation purification method used is first use recombinant human interleukin--2 9 varient in strong cation type ion exchange chromatography separation and Culture liquid, then obtains recombinant human interleukin--2 9 varient of purifying with gel permeation chromatography.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113150107A (en) * | 2020-05-27 | 2021-07-23 | 杭州先为达生物科技有限公司 | Interleukin 29 mutant protein |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102559737A (en) * | 2011-12-13 | 2012-07-11 | 江南大学 | Human interleukin 29 variant and preparation method thereof |
| CN103224951A (en) * | 2013-01-25 | 2013-07-31 | 江南大学 | Human interleukin29 mature peptide mutant (hIL-29G) with lysine produced by site-directed mutagenesis of 35th arginine and preparation method thereof |
| CN103224952A (en) * | 2013-01-25 | 2013-07-31 | 江南大学 | Human interleukin29 mature peptide mutant (hIL-29Z) with arginine and lysine produced respectively by site-directed mutagenesis of 33th lysine and 35th arginine and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102559737A (en) * | 2011-12-13 | 2012-07-11 | 江南大学 | Human interleukin 29 variant and preparation method thereof |
| CN103224951A (en) * | 2013-01-25 | 2013-07-31 | 江南大学 | Human interleukin29 mature peptide mutant (hIL-29G) with lysine produced by site-directed mutagenesis of 35th arginine and preparation method thereof |
| CN103224952A (en) * | 2013-01-25 | 2013-07-31 | 江南大学 | Human interleukin29 mature peptide mutant (hIL-29Z) with arginine and lysine produced respectively by site-directed mutagenesis of 33th lysine and 35th arginine and preparation method thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113150107A (en) * | 2020-05-27 | 2021-07-23 | 杭州先为达生物科技有限公司 | Interleukin 29 mutant protein |
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