CN1626554A - Interfusion protein between human serum albumin and interleukin, and encoding genes - Google Patents
Interfusion protein between human serum albumin and interleukin, and encoding genes Download PDFInfo
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- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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Abstract
A fusion protein of human serum albumin and internaleukin 2 for elongating the half life of interleukin 2 in human serum including the first region of amino acid residue sequence with at least 85 of homology to SEQ ID No.1 and its second region with at least 85% of homology to SEQ ID No.2. It can be used to prepare the medicines for treating the diseases associated with human interleukin 2 or its excess expression.
Description
Technical field
The present invention relates to a kind of fusion rotein and encoding gene thereof and application, particularly relate to fusion rotein and the encoding gene and the application of a kind of human serum albumin and interleukin II.
Background technology
Serum albumin is the important composition of blood plasma, also is the carrier of many castle's intrinsic factors and external source medicine, is difficult for seeing through renal glomerulus under the normal circumstances.Human serum albumin (HAS) is a kind of protein of being made up of 585 amino-acid residues (sequence 1 in the sequence table) (A.Dugaiczyk et al., PNAS, 1982 79:71-75), and its molecular weight is about 66.5KD, reaches plasma half-life more than 2 weeks.
HSA is with a kind of signal peptide of 18 amino-acid residues and former peptide form synthetic of 6 propetides of containing in cell, and signal peptide and propetide are cut in transhipment and secretion process.Human serum albumin is successfully expressed (EP330451 and EP361991) in multiple host.
Interleukin II (IL-2) is the 15.5kD glycoprotein that T cell and NK cell produce, and plays an important role in the immunne response of body.(people) interleukin II ((h) IL-2) is formed (sequence 2 in the sequence table) by 133 amino-acid residues, an intrachain disulfide bond is arranged, with a halfcystine that is positioned at maturation protein the 125th amino acids residue, this halfcystine easily with other two halfcystines formation mispairing disulfide linkage, the unusual paired IL-2 of disulfide linkage does not have activity.With this cysteine mutation is that Serine or L-Ala can be avoided this problem, and does not change its activity.IL-2 mainly acts on immunocyte, comprises T cell, large granular lymphocyte, monocyte, B cell and oligodendroglia, promotes cell proliferation and secrete cytokines.In vivo, IL-2 has effects such as antitumor, anti-microbial infection and immunomodulatory.Use separately or share in clinical, renal cell carcinoma, metastasis melanin tumor and lymphoma etc. are had certain therapeutic action with IFN-α, monoclonal antibody, LAK cell etc., also effective in cure to colorectal carcinoma, can also improve tumour patient to put, the tolerance of chemotherapy.Clinically it is widely used at present the treatment of tumour, viral hepatitis etc., evident in efficacy.But the general course of disease of above-mentioned disease is longer, thereby needs life-time service IL-2, and only be about 2 hours the plasma half-life of IL-2, need heavy dose of frequent injection for keeping drug effect, this has not only improved the treatment cost greatly, and has increased patient's misery, has increased side reaction.
The innovation and creation content
But the human serum albumin and interleukins 2 fusion protein and the encoding gene thereof that the purpose of this invention is to provide a kind of plasma half-life of prolonged human interleukin II.
The fusion rotein of human serum albumin provided by the present invention and interleukin II comprises and SEQ ID №: 1 have at least 85% sequence homology amino acid residue sequence first district and with sequence table in SEQ ID №: 2 have second district of the amino acid residue sequence of at least 85% sequence homology.
SEQ ID № in the sequence table: the 1st, the human serum albumin of forming by 585 amino-acid residues; SEQ ID № in the sequence table: the 2nd, the human interleukin-12 of forming by 133 amino-acid residues.
Wherein, the fusion rotein of human serum albumin and interleukin II preferably comprise with sequence table in SEQ ID №: 1 have at least 95% sequence homology amino acid residue sequence first district and with sequence table in SEQ ID №: 2 have second district of the amino acid residue sequence of at least 95% sequence homology; Most preferably comprise SEQ ID №: SEQ ID № in first district of 1 amino acid residue sequence and the sequence table: second district of 2 amino acid residue sequence, or the function equivalent in above-mentioned two districts, described function equivalent is under the prerequisite that does not change described fusion rotein characteristic, the polypeptide that replacement, disappearance or the interpolation of the indivedual amino-acid residues of described fusion rotein obtained.
Wherein, described replacement can occur in SEQ ID № in the sequence table: 2 from N-terminal the 125th halfcystine, can be replaced by Serine or L-Ala.
In fusion rotein of the present invention, can be with sequence table in SEQ ID №: 1 homologous, first district is positioned at the N-end of fusion rotein, and SEQ ID № in the sequence table: 2 homologous, second district is positioned at the C-end of fusion rotein; Also can be with sequence table in SEQ ID №: 2 homologous, second district is positioned at the N-end of fusion rotein, and SEQ ID № in the sequence table: 1 homologous, first district is positioned at the C-end of fusion rotein, but the expression of the gene of the previous case in yeast is higher.
In order to make bigger interval is arranged between fusion rotein two portions, make human interleukin-12 part most possibly with the interleukin II receptors bind, SEQ ID № in the described and sequence table: SEQ ID № in 1 homologous, first district and the sequence table: 2 homologous are provided with connection peptides between second district, and the general formula of described connection peptides is [GlyGlyGlyGlySer]
n, n is the integer of 1-10; Wherein, preferably n is the integer of 1-3.
The fusion rotein of above-mentioned human serum albumin and interleukin II is preferably has SEQ ID № in the sequence table: the protein of 4 amino acid residue sequences.
SEQ ID № in the sequence table: the 4th, the protein of forming by 723 amino acid residue sequences, wherein from N-terminal the 1st the-the 585th the sero-abluminous encoding sequence of behaving, from N-terminal the 586th the-the 590th be the encoding sequence GlyGlyGlyGlySer of connection peptides, be the encoding sequence of human interleukin-12 from N-terminal the 591st the-the 723rd.
The encoding gene of above-mentioned fusion rotein also belongs to protection scope of the present invention; wherein; preferably by SEQ ID № in the sequence table: the encoding gene of the human serum albumin that 4 amino acid residue sequence is formed and the fusion rotein of interleukin II, it is one of following nucleotide sequences:
1) SEQ ID № in the sequence table: 3 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of april protein sequence;
3) with sequence table in SEQ ID №: 3 dna sequence dnas that limit have 80% above homology, and the identical function protein DNA sequence of encoding.
SEQ ID № in the sequence table: 3 dna sequence dna is by 2253 based compositions, and the reading frame of this gene is from 5 ' end the 9th to the 2253rd bit base.
The recombinant expression vector and the clone that contain above-mentioned encoding gene all belong to protection scope of the present invention.Described recombinant expression vector can be pHIL-D2HSAIL-2, pHIL-D2HSAIL-20, pHIL-D2HSAIL-22, pHIL-D2HSAIL-23, pPIC9-IL-2-HSA plasmid etc.; Institute's clone can be bacterium, yeast, zooblast or the vegetable cell that contains above-mentioned encoding gene; Wherein preferably yeast, more preferably pichia spp, most preferably pichia spp GS115.
The natural polymorphism that exists of human serum albumin, the human serum albumin part in the fusion rotein of the present invention also comprises these multiformities.
The polynucleotide of the polynucleotide of coding HSA and coding hIL-2 can be used the known method in this area, as PCR (Science.239:487-491 such as Saiki, 1988), the acquisitions such as method in the method for RT-PCR method, synthetic and structure screening cDNA library, as pcr template be used for the mRNA in construction cDNA library or cDNA can derive from any tissue that contains corresponding mRNA or cDNA, cell, and library etc., as obtaining from people liver tire cDNA library (available from Clontech Laboratories Inc.USA).Also the codon that can select for use the host to have a preference for during synthetic, the expression that so often can improve product can be obtained with artificial synthetic method.As required, available method well known in the art is suddenlyd change, lacks, inserts and is connected with other polynucleotide polynucleotide etc.The fusion of the polynucleotide of coding HSA and coding hIL-2, keeping under the prerequisite that reading frame is constant separately, can use the known the whole bag of tricks in this area, as method by PCR, the restriction enzyme enzyme recognition site is introduced in both sides at encoding sequence, cut the generation sticky end by enzyme, coding HSA is connected with dna ligase with the sticky end of the polynucleotide of coding hIL-2, thereby obtains the gene of encoding fusion protein.If desired, can introduce polynucleotide in the both sides of encoding fusion protein gene of the present invention, the polynucleotide of introducing can restricted property restriction endonuclease recognition site.Available method well known in the art will contain the nucleic acid clone of encoding fusion protein sequence in various expression vectors.The molecular cloning process of used standard is seen (J. Sa nurse Brooker etc., " molecular cloning experiment guide " second edition, Science Press, 1995.) such as J. Sa nurse Brookers.
Expression vector and its corresponding host can be buied from company, as Yeast expression carrier pHIL-D
2, pPIC9, pHIL-S
1(Invitrogen Corp.San Diego.California.USA), animal cell expression carrier pSVK3, pMSG (Amersham Pharmacia Biotech Inc.USA) etc.Preferable methods is that nucleic acid clone with coding fusion rotein of the present invention or polypeptide is to Yeast expression carrier pHIL-D
2, pPIC9 etc., these plasmids are yeast integrative plasmid, have the His4 selected marker.Alcohol oxidase operon (AOX1) 5 ' sequence and 3 ' sequence are used to make things convenient for encoding gene to be integrated into the expression of yeast chromosomal and control encoding gene.These plasmids and corresponding host bacterium etc. can get from Invitrogen Corp.San Diego.California.USA structure, and preferred promotor is AOX1.
The host who expresses fusion rotein of the present invention can be yeast, mammalian cell, bacterium, animal, plant etc.The fusion rotein or the polypeptide of expressing may reside in the host cell, also can be that secretion is come out from the host, and preferred phraseology is that secretion is come out from the host.Secrete used signal peptide, the signal peptide of preferably natural human serum albumin, or yeast MF signal peptide, or the analogue of these two kinds of signal peptides.The signal peptide of the human serum albumin that preferred usefulness is natural, the expressing fusion protein level is higher during with this signal peptide.Fusion rotein or polypeptide also can be without signal peptides, and express with soluble form in the born of the same parents in yeast.The nucleic acid of encoding fusion protein can be inserted into host chromosome, or exists with the free plasmid form.
Required nucleic acid is imported host cell can use usual method, as: electroporation prepares competent spheroplast etc.Positive cell promptly contains the cell of DNA recombinant chou of the present invention, can be identified by the technology that people know, and through collecting and cracking, extracts DNA as cell, and PCR method is identified then; In the cell culture supernatant or the albumen in the cytoclasis liquid can be with the antibody test of anti-HSA or anti-IL-2.
Can contain the host of DNA recombinant vectors of the present invention by cultivation,, produce fusion rotein of the present invention as recombination yeast, recombinant mammalian cells, recombinant bacteria, genetically modified animals and plants etc.Concrete cultural method can be preferably bio-reactor with shaking bottle or bio-reactor etc. during production.Used substratum should be able to provide thalline (or cell) growth and product to express required nutritive substance, should comprise nitrogenous source, carbon source, pH buffer composition etc., and culture medium prescription generally should obtain by test according to different Objects of Development.Cultivation can be divided into two stages, and the fs is mainly used in the growth of thalline (or cell), and subordinate phase is mainly used in synthetic product.
Can separate with the method for various protein isolates, purifying fusion rotein of the present invention, as saltout, the combination of technology such as precipitation, ultrafiltration, liquid chromatography (LC) and these technology.Wherein liquid chromatography (LC) can be used gel exclusion, affine, ion-exchange, chromatographic technique such as hydrophobic, anti-phase.
Fusion rotein of the present invention can have various derivatives, and these derivatives can be but be not limited to its multi-form salt, modified outcome etc., as modifying on the amino of polypeptide, carboxyl, hydroxyl, sulfydryl again; Used modifier can but be not limited to polyoxyethylene glycol, dextran etc.
Fusion rotein of the present invention and derivative thereof can use separately, preferably form pharmaceutical preparation with one or more pharmaceutically acceptable carriers.Pharmaceutical carrier generally should be compatible with fusion rotein and can not be harmful to receptor autophosphorylation, and typical carrier is water, salt solution, carbohydrate, alcohols or amino acid, and their need aseptic and do not have pyrogeneous substance.Pharmaceutical preparation can unitary dose form exist, preferred dosage unit includes the daily dosage of daily dosage or unit or its suitable Asia branch of fusion rotein, and can prepare by any methods known in the art.These methods comprise fusion rotein and one or more ancillary component blended steps.Preferred drug substances comprises that aqueous liquid preparation and water content are lower than 10% or water-free freeze-dried preparation (freeze-dried preparation adds aseptic, pyrogen-free liquid carrier before using, as water for injection with liquid preparation lyophilize preparation).These preparations can contain buffer reagent, salt, and small molecules carbohydrates etc. make the perviousness of medicine equate with perviousness in the acceptor blood or similar.Pharmaceutical preparation can be present in the container of dosage unit or multiple doses, as the peace bottle of sealing, in cillin bottle or the tubule.
Fusion rotein of the present invention and derivative thereof or its pharmaceutical composition can be by any known methods, comprise injection (as subcutaneous or muscle), venoclysis, transdermal, suction, method administration such as oral.Preferable methods is venoclysis or drug administration by injection.Therapeutic modality is included in and uses single dose or compound dosage in for some time.
Fusion rotein of the present invention both can keep the activity that activates the human interleukin-12 acceptor, agonist as this receptor, make human interleukin-12 class medicine, also can only keep and this receptor bonded activity, as the inhibitor of this receptor, make the medicine of some and human interleukin-12 or its acceptor overexpression diseases related of treatment.Fusion rotein of the present invention and derivative thereof or its pharmaceutical composition, can use separately or share the disease that is used for the treatment of various available IL-2 treatments as IL-2 class medicine with IFN-α, monoclonal antibody, LAK cell etc., these diseases can be tumour, infected by microbes etc., as the treatment of renal cell carcinoma, melanoma, lymphoma colorectal carcinoma, viral hepatitis etc.Can be used for treating some and interleukin II or its acceptor overexpression diseases associated as the IL-2 acceptor inhibitor, as some leukemia etc.Using dosage can calculate with respect to tiring of human interleukin-12 (hIL-2) by fusion rotein, considers the transformation period that fusion rotein prolongs with respect to natural hIL-2 simultaneously.Typical amounts is 6.25 * 10
4-6 * 10
5IU/kg.In the fusion rotein that forms by total length HSA and total length h IL-2, mean at once mutually according to mole number and to use the greater weight fusion rotein, but frequency of utilization can reduce, as Wednesday time, biweekly, weekly or still less.
The present invention is with the human serum albumin molecule, or have analogue or the fragment and a human interleukin-12 of at least 85% sequence homology with it, or the analogue or the fragment that have at least 85% sequence homology with it merge, formed fusion rotein had both kept the activity with the human interleukin-12 receptors bind, simultaneously compare the prolongation of having got back its plasma half-life with human interleukin-12.Experiment showed, that fusion rotein of the present invention compares with rhIL-2, the transformation period in mice plasma obviously prolongs.
The present invention will be further described below in conjunction with the drawings and specific embodiments.
Description of drawings
Fig. 1 is the building process synoptic diagram of pHIL-D2HSAIL-2 plasmid
Fig. 2 is the building process synoptic diagram of pD3HSAIL-20 plasmid
Fig. 3 is the building process synoptic diagram of pD3HSAIL-22 plasmid
Fig. 4 is a pPIC9-IL-2-HSA plasmid construction process synoptic diagram
Fig. 5 is that the SDS-PAGE of the fusion rotein HSA/IL-2 of purifying analyzes
Fig. 6 is fusion rotein HSA/IL-2 enzyme immunoassay
Fig. 7 is the stimulating activity of fusion rotein HSA/IL-2 to the CTLL-2 cell
Embodiment
The clone of the clone of embodiment 1, hIL-2cDNA and HSA cDNA
1, the clone of hIL-2cDNA
(1) preparation of the total RNA of human peripheral leucocytes
Vein extracts normal adult human blood 5mL, heparin sodium anti-freezing.Behind blood usefulness RPMI 1640 substratum doubling dilutions, add lymphocyte separation medium, with the centrifugal 20min of 2000r/min.Draw leukocytic cream, wash cell 3 times and cell count is adjusted into 1 * 10 with RPMI 1640 substratum that contain the 100ml/L calf serum
9/ L, adding final concentration is the PHA of 60mg/L, in 37 ℃, 50ml/L CO
2Activate 5h in the incubator.Collecting cell is used the total RNA extraction reagent box, extracts total RNA and be used for reverse transcription reaction from cell.
(2) clone of hIL-2cDNA
Method according to the RT-PCR test kit is carried out reverse transcription reaction.Carry out PCR with the reverse transcription product that obtains as template and obtain hIL-2cDNA, used primer I L-2a and IL-2b are synthetic with the oligonucleotide synthesizer.
IL-2a：5’ATGGATCC?GCA?CCT?ACT?TCA?AGT?TCT?ACA?AAG?AAA?AC?3’
IL-2b：5’GAGAATTC?TTA?AGT?TAG?TGT?TGA?GAT?GAT?GCT?TTG?AGA?AAA?GGT?AATCCA3’
PCR method is:
Add 1 μ l human lymphocyte cDNA in the 100 μ l reaction systems, the IL-2a of 20 μ mol/L, each 2 μ l of IL-2b primer, the dNTP 8 μ l of 2mmol/L, 10 * reaction buffer, 10 μ l, pfu archaeal dna polymerase 5U, (dNTP, reaction buffer, pfu archaeal dna polymerase are Shanghai biotechnology Services Co., Ltd product).With 9600 PCR instrument of Perk-Elmer company, the PCR condition is 94 ℃ of sex change 30 seconds, anneals 1 minute for 52 ℃, and 72 ℃ were extended 30 seconds, and circulated 32 times.Show the band of an expection big or small (0.4kb) by the gel electrophoresis analysis reactant, the PCR product reclaims the purification kit purifying with the PCR product and reclaims.Cut with BamH I and EcoR I enzyme, behind the agarose gel electrophoresis purifying, be cloned into on the pUC19 plasmid of BamH I/EcoR I enzymolysis (the pfu enzyme in the experiment, restriction endonuclease, ligase enzyme, pUC19 plasmid, test kit etc. are available from vast Tyke biological gene technology company limited of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and Beijing), form pUC-IL-2.BamH I-EcoR I district dna sequencing result shows that this PCR product sequence is identical with the hIL-2 sequence, has just added BamHI site and EcoR I site respectively at its 5 ' end and 3 ' end.And before 3 '-end EcoR I site, added terminator codon.
2, the clone of HSA cDNA
Obtain to have the HSA cDNA of signal peptide and propeptide code sequence from people liver tire cDNA library (available from Clontech Laboratories Inc.USA) with PCR method, used primer HSA1 and HSA2 are synthetic with the oligonucleotide synthesizer.
HSA1:5 ' GC
TTCGAAACCATGAAGTGGGTAACCTTTATTTCCCT3 ' (base sequence of line is a restriction enzyme site)
HSA2:5 ' TA
GGATCCACCACCACCAAGGCCTAAGGCAGCTTGACTTGC3 ' (base sequence of line is a restriction enzyme site)
The PCR condition is: in the 100 μ l reaction systems, add 1 μ l people liver tire cDNA library, each 3 μ l of the HSA1 of 20umol/L and HSA2 primer, the dNTP of 2mmol/L, 10 μ l, 10 * reaction buffer, 10 μ l, TaqplusI archaeal dna polymerase 5U.With 9600 PCR instrument of Perk-Elmer company, the PCR condition is 94 ℃ of sex change 1 minute, anneals 1 minute for 52 ℃, and 72 ℃ were extended 3 minutes, and after 35 circulations, 72 ℃ were extended 10 minutes again.The band that shows an expection size (1.8KD) by the gel electrophoresis analysis reactant, PCR product electrophoresis purifying reclaims the object tape (the TaqplusI archaeal dna polymerase in the experiment, restriction endonuclease, ligase enzyme, test kit etc. are available from vast Tyke biological gene technology company limited of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and Beijing) that test kit reclaims the about 1.8KD of purifying with dna fragmentation.Get the HSA cDNA 6 μ l of purifying, add 1 μ l pGEM-T carrier, 8 μ l2 * damping fluids, 1 μ l T4DNA ligase enzyme (pGEM-T carrier, damping fluid and enzyme are Promega Corp.USA product), 15 ℃ of reactions are spent the night, and transform JM109 competent cell (cell is available from vast Tyke, Beijing biological gene technology company limited).Transformed bacteria on the LB agar plate that contains x-gal and IPTG and penbritin (50ug/ml), 37 ℃ of overnight incubation.The picking white colony is seeded to the LB substratum that 3ml contains penbritin (50ug/ml), 37 ℃ of overnight incubation, with ordinary method extracting plasmid, cut with the PstI enzyme, (on the carrier He on the HSA cDNA PstI restriction enzyme site being arranged respectively), positive colony is identified in electrophoretic analysis.Check order respectively from the multiple clone site two ends of pGEM-T carrier, and synthetic two sequencing primers:
HSA3：5’GCCAGAAGACATCCTTAC3’
HSA4：5’AGTTGGAGTAGACACTTG3’
From the then order-checking of middle part, two ends, finish the order-checking of HSA cDNA total length.Obtained to contain and the consistent clone of natural HSA cDNA sequence, JM109 (pGEMT-HSA), Ke Long HSA contains its natural signals peptide and preceding peptide moiety here, so that when expressing, directly utilize its signal peptide and propetide to be used for as secretion signal.
For with HSA and hIL-2 with the fusion rotein form from the pichia spp secreting, expressing, as shown in Figure 1, select the pHIL-D2 plasmid as carrier (Invitrogen Corp.USA).Between this carrier A OX promotor downstream NspV and EcoRI site, insert the HSA/IL-2 gene, because of two NspV sites are arranged on the pHIL-D2 carrier, operation for convenience, at first the pHIL-D2 carrier is cut into 3 sections with the ClaI/ScaI enzyme, reclaim the 4kb fragment, self connect back called after pD3 carrier.The pD3 carrier is cut with NspV/EcoRI, reclaim linearized vector, the pUC-IL-2 that embodiment 1 is made up cuts with BamHI/EcoRI, reclaim the fragment that contains IL-2cDNA of about 0.5kb, the pGEMT-HSA that embodiment 1 is made up cuts with NspV/BamHI, reclaim the fragment that contains HSAcDNA of about 1.8kb, with linearizing pD3 carrier with contain HSAcDNA, the fragment of IL-2cDNA connects with the catalysis of T4 ligase enzyme, transform JM109, select positive colony, cut evaluation with NspV/EcoRI and NspV/BamHI enzyme respectively, the acquisition structure is correct, has the positive colony JM109 (pD3HSAIL-2) that coding contains the HSA-IL-2 antigen-4 fusion protein gene of GlyGlyGlyGlySer joint peptide.PD3HSA IL-2 plasmid is cut with the ClaI/ScaI enzyme, and electrophoresis reclaims linearizing 6.3kb fragment, and the pHIL-D2 empty carrier cuts back to close the fragment of about 4.2kb with ClaI/ScaI.T
4Ligase enzyme catalysis connects the 6.3kb fragment of pD3HSAIL-2 and the 4.2kb fragment of pHIL-D2, after the connection product transforms JM109, is laid on the LB agar plate that contains penbritin (50ug/ml), select positive colony, the extracting plasmid, enzyme is cut evaluation, obtains clone JM109 (pHIL-D2HSAIL-2).The about 10 μ g of this plasmid of extracting cut with the NotI enzyme again.Transform pichia spp GS115 (the yeast expression test kit is provided by InvitrogenCorp.USA) after the linearizing, the GS115 after the conversion is laid on the RDB agar plate (1mol/L sorbyl alcohol, 1% glucose, 4 * 10 that contain sorbyl alcohol
-5The % vitamin H, 1.34% no amino acid yeast nitrogen (YNB, Difco Corp.), 0.005% amino acid mixing liquid (L-glutamic acid, methionine(Met), Methionin, leucine, Isoleucine each 0.005%)), cultivate 1-2 weeks, picking His for 37 ℃
+The clone.Be seeded to 25mlBMGY substratum (100mmol/L potassiumphosphate, pH6.0,4 * 10 are housed
-5% vitamin H, 1.34% no amino acid yeast nitrogen, 1% glycerine) the 300ml triangular flask, cultivated 48 hours, and added methyl alcohol 0.5%/24h, inducing culture 4 days, centrifuging and taking supernatant, filtration sterilization for 250 rev/mins 30 ℃.Measure the activity of IL-2 in the culture supernatant with CTLL-2.The method of determination of activity is referring to " Chinese biological goods rules " 2000 editions (the Chinese biological standard of articles council compiles, Chemical Industry Press 2000.6) P396-398.With the CTLL-2 cell cultures in 1640 substratum that contain 10% calf serum and 400-800IU/ml rhIL-2 (Gibco BRL USA).Go down to posterity weekly 3 times, 1640 substratum that contain 10% calf serum with preceding usefulness are washed cell 4 times, are diluted to 5 * 10
5Cell/ml.On 96 orifice plates, sample is done suitably dilution with 1640 substratum that contain 10% calf serum, every hole 50 μ l, and every hole adds cell 50 μ l, 5%CO again
2Cultivate after 18-24 hour for 37 ℃, add MTT solution (tetrazolium bromide solution, be made into the solution of 5.0mg/ml with phosphate buffered saline buffer, through 0.22 μ m membrane filtration degerming) 20 μ l, continue to cultivate 4-6 hour, add 20%SDS (sodium laurylsulfonate) solution, hatched 18-24 hour for 37 ℃, join the absorbance that instrument is measured each sample well, select the high clone of absorbance and be high-expression clone with enzyme.The product that obtains is analyzed through SDS-PAGE, and molecular weight is correct, and enzyme immunoassay proves that it has the characteristic of serum albumin and human interleukin-12.The slip-knot of CTLL-2 cell survey simultaneously shows that really product has the biological activity of hIL-2.
Embodiment 3, HSA/IL-2 (Ser
125) Expression of Fusion Protein
Present embodiment provides a kind of IL-2 maturation protein HSA/IL-2 (Ser that the 125th cysteine mutation is Serine
125) preparation method of fusion rotein: use the method identical to make up pHIL-D2HSAIL-2 with embodiment 1 and 2, be template, obtain hIL-2 (Ser with PCR
125) cDNA, used primer I L-2a and IL-2 (Ser125): synthetic with the oligonucleotide synthesizer.
IL-2a:AT
GGATCCGCA CCT ACT TCA AGT TCT ACA AAG AAA AC (base sequence of line is a restriction enzyme site)
IL-2 (Ser125): 5 ' GA
GAATTCTTATGTCAGTGTTGAGATGATGCTTTGACTAAAGGTAATCCATCTGTTCAG (base sequence of line is a restriction enzyme site)
PCR method is:
The pD2HSA IL-2 that adds 100 times of dilutions of 1 μ l in the 100 μ l reaction systems, each 3 μ l of the IL-2a of 20 μ mol/L, IL-2 (Ser125) primer, the dNTP 10 μ l of 2mmol/L, 10 * reaction buffer, 10 μ l, pfu archaeal dna polymerase 5U, (dNTP, reaction buffer, pfu archaeal dna polymerase are Shanghai biotechnology Services Co., Ltd product).With 9600 PCR instrument of Perk-Elmer company, the PCR condition is 94 ℃ of sex change 1 minute, anneals 30 seconds for 52 ℃, and 72 ℃ were extended 1 minute, and circulated 25 times.Show the band of an expection big or small (0.4kb) by the gel electrophoresis analysis reactant, the PCR product reclaims the purification kit purifying with the PCR product and reclaims.Cut with BamH I and EcoRI enzyme, the agarose gel electrophoresis purifying, be connected (the pfu enzyme in the experiment, restriction endonuclease, ligase enzyme, pUC19 plasmid, test kit etc. are available from vast Tyke biological gene technology company limited of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and Beijing) with the pD2HSAIL-2 plasmid of BamHI/EcoRI enzymolysis excision IL-2 encoding sequence, form pD2HSAIL-2 (Ser125).The dna sequencing result shows that sudden change is correct.PD2HSA IL-2 (Ser125) cuts with the NotI enzyme, after the linearizing, uses the method identical with embodiment 2 to make up and transforms pichia spp GS115, and screening efficiently expresses the engineering yeast of pD2HSA/IL-2 (Ser125) fusion rotein.
Embodiment 4, IL-2/HSA Expression of Fusion Protein
PPIC9-IL-2-HSA plasmid construction process specifically can comprise following process as shown in Figure 4:
Obtain hIL-2cDNA from liver tire cDNA library with the PCR method similar to embodiment 1, used primer changes IL-2c and IL-2d into, and primer is synthetic with the oligonucleotide synthesizer.
IL2c:5 ' AT
GAATTC CTCCGAGAAA AGA GCA CCT ACT TCA AGT TCT ACA AAG AAA AC3 ' (base sequence of line is a restriction enzyme site)
IL-2d:5 ' TA
GGATCCACC ACC GCC AGT TAG TGT TGA GAT GAT GCT TTG AGA AAA GGTAAT CCA (base sequence of line is a restriction enzyme site)
Behind the pcr amplification, introduce an EcorI site and an XhoI site at 5 ' of cDNA-end, the KEX2 restriction enzyme site that induced one in downstream, XhoI site simultaneously, in order to excise the signal peptide of the fusion rotein of expressing, 3 ' end is introduced the BamHI site.Behind the PCR product electrophoresis purifying, be cloned into and use BamH I/ecoRI, on the pUC19 of enzymolysis (vast Tyke, Beijing biological gene technology company limited) plasmid, form pUC-IL-2.The dna sequencing result shows that this cloned sequence is identical with the hIL-2 sequence, just EcoRI site, XhoI site, KEX2 restriction enzyme site have been added at its 5 '-end, remove terminator codon with 3 ' end, added the encoding sequence GGTGGTGGTGGATCC of flexible joint GlyGlyGlyGlySer.
Method with PCR obtains HSA cDNA from people liver tire cDNA library, used primer HSA5 and HSA6 are synthetic with the oligonucleotide synthesizer:
HSA5：5’-TAGGATCC?GAT?GCA?CAC?AAG?AGT?GAG?GTT-3’
HSA6：5’-ATGAATTC?TTAAAGGCCTAAGGCAGCTTGACTTGC-3’
PCR method, is cloned into and is used BamHI/SalI behind the PCR product electrophoresis purifying with embodiment 1, on the pUC19 of enzymolysis (vast Tyke, Beijing biological gene technology company limited) plasmid, forms pUCHSA2.The dna sequencing result shows that this cloned sequence is identical with the encoding sequence of HSA mature peptide, just adds the SalI site after its 5 '-end has added BamHI site and 3 ' end terminator codon.
Select the pPIC9 plasmid as expression vector (Invitrogen Corp.USA).Between this carrier A OX promotor downstream XhoI and EcoRI site, insert the IL-2/HSA gene.The pUC19-IL-2 that makes up is above cut with the BamHI/EcoRI enzyme, reclaim the fragment that contains IL-2cDNA of about 0.4kb; PUCHSA2 is cut with the BamHI/SalI enzyme, reclaim the fragment that contains HSA cDNA of about 1.8kb; The pPIC9 plasmid is cut with the SalI/EcoRI enzyme, reclaimed the linearizing fragment, these three fragment T
4Dna ligase connects, transform JM109, select positive colony, cut evaluation with SalI/BamHI and BamHI/EcoRI enzyme respectively, the acquisition structure is correct, has the positive colony JM109 (pPIC9 IL-2-HSA) of the fusion rotein IL-2-HSA gene of joint peptide GlyGlyGlyGlySer.After pPIC9 IL-2-HSA plasmid is used the Bg/II linearization for enzyme restriction, transform pichia spp GS115 (method that Invitrogen Corp.USA company yeast expression test kit provides), GS115 after the conversion, be laid on the RDB agar plate (prescription is with embodiment 2) that contains sorbyl alcohol, cultivate 1-2 week, picking His for 37 ℃
+The clone.Shake-flask culture and expression strain screening method are with embodiment 2, and the cultivation of engineering zymic, product purification, IL-2 determination of activity, HSA antigenic determination method are with embodiment 2.The result shows that the IL-2/HSA fusion protein molecule amount of expression is correct, and having stimulates CTlL-2 cell proliferation, i.e. the antigenicity of the activity of hIL-2 and HSA, but expression level is lower than the HSA/IL-2 of embodiment 2.
Embodiment 5, with the Expression of Fusion Protein of different joints
The building process of plasmid pD3HSAIL-20 as shown in Figure 2, with the plasmid pD3HSAIL-2 that makes up among the embodiment 2, use the BamHI/StuI double digestion, reclaim big fragment, handle with the Semen Phaseoli radiati Germinatus nuclease, the excision protruding terminus, T4DNA ligase flush end connects, and transforms the JM109 bacterium, selects positive colony, enzyme is cut evaluation, obtains to have in HSAcDNA terminal deletion CTT (leucine) back the plasmid pD3HSAIL-20 with the direct fusion gene of IL-2 cDNA.
Equally, can add multiple flexible joint at HSA and IL-2, as [GlyGlyGlyGlySer]
n, n can be 1-10.As when the n=2, can synthesize oligonucleotide:
L2a：5’CCTTGGTGGTGGCGGTTCCGGCGGTGGTG?3’
L2b：5’GATCCACCACCGCCGGAACCGCCACCACCAAGG3’
The building process of pD3HSAIL-22 plasmid as shown in Figure 3, with the plasmid pD3HSAIL-2 that makes up among the embodiment 2, use the BamHI/StuI double digestion, reclaim big fragment, oligonucleotide L2a is connected with the big fragment that reclaims with L2b annealing back, transforms JM109, the picking positive colony, enzyme is cut and is identified and sequential analysis, obtains to have to insert coding [GlyGlyGlyGlySer] between HSAcDNA end and IL-2cDNA
2The plasmid pD3HSAIL-22 of the oligonucleotide of (being GlyGlyGlyGlySerGlyGlyGlyGlySer) flexible joint.
When n=3, can synthesize oligonucleotide:
L3a：5’CCTTGGTGGTGGCGGTTCCGGCGGTGGTGGCTCCGGTGGCGGCG3’
L3b：5’GATCCGCCGCCACCGGAGCCACCACCGCCGGAACCGCCACCACCAAGG3’
With the plasmid D3HSAIL-2 that makes up among the embodiment 2, use the BamHI/StuI double digestion, reclaim big fragment, oligonucleotide L3a is connected with the big fragment that reclaims with L3b annealing back, transform JM109, the picking positive colony, enzyme is cut evaluation, obtains to have between HSAcDNA end and GM-CSF cDNA to insert coding [GlyGlyGlyGlySer]
3The plasmid pD3HSAIL-23 of the oligonucleotide of (being GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) flexible joint.Available similar methods is transformed the pPIC9 IL-2-HSA plasmid that embodiment 4 makes up, to insert different joints between IL-2 and HSA.
Plasmid with above structure, use the method identical to make up corresponding plasmid pHIL-D2HSAIL-20, pHIL-D2HSAIL-22, pHIL-D2HSAIL-23 respectively as pD3HSAIL-20, pD3HSAIL-22, pD3HSAIL-23 with embodiment 2, and transform pichia spp GS115, insert [GlyGlyGlyGlySer] between acquisition is expressed behind the HSA terminal deletion leucine and IL-2 directly merges HSA/IL-20 fusion rotein, HSA and IL-2
2The fusion rotein HSA/IL-22 of flexible joint, and HSA and IL-2 insertion [GlyGlyGlyGlySer]
3The fusion rotein HSA/IL-23 of flexible joint etc.The product S DS-PAGE that obtains analyzes, and molecular weight is correct, and enzyme immunoassay proves that it has the characteristic of serum albumin and human interleukin-12.The slip-knot of CTLL-2 cell survey simultaneously shows that really product has the biological activity of hIL-2.
The purifying of embodiment 6, the fermentation of engineering zymic and fusion rotein
The engineering yeast fermentation of the expression HSA/IL-2 fusion rotein that makes up with embodiment 2 and the purifying of fusion rotein are example, and the purifying of other engineering zymic fermentation and fusion rotein can be used same or analogous method.
Engineering yeast-inoculated 100mlYPD substratum (yeast extract 10g/L, Tryptones 20g/L, glucose 10g/L), 30 ℃ of shaking tables are cultivated 24h for 280 rev/mins.Be seeded to the 5L fermentor tank (B.Braun Corp.Germany) that the 2.5L basic medium is housed, wherein the compound method of basic medium is: strong phosphoric acid 3.5ml/L, CaSO
4.2H
2O 0.15g/L, K
2SO
42.4g/L, MgSO
4.7H
2O 1.95g/L, KOH 0.65g/L, 121 ℃ of autoclavings 30 minutes add 30ml/L glycerine (independent 121 ℃ of autoclavings 30 minutes) again, and (prescription is CuSO to 1ml/L PTM
4.5H
2O 6.0g/L, CoCl
2.6H
2O 3.0g/L, MnSO
4.H
2O 3.0g/L, H
3BO
30.02g/L, FeSO
4.7H
2O 65.0g/L NaMoO
4.2H
2O 0.2g/L, ZnSO
4.7H
2O 20.0g/L, KI 0.1g/L, dense H
2SO
45ml/L, 121 ℃ of autoclavings 30 minutes), 0.5ml/L 0.02% vitamin H (filtration sterilization).With ammoniacal liquor medium pH is transferred to 5.8 before the inoculation.The fermenting process controlled temperature is 30 ℃, and dissolved oxygen is all the time greater than 20% saturation ratio, and the pH value is not higher than 6.0, after being cultured to glycerine and exhausting, begins to flow glycerol adding (50% glycerine adds 12ml/LPTM, the 2ml/L0.02% vitamin H), continues to cultivate, to density OD
600Value is about at 150 o'clock, begins to add methyl alcohol (analytical pure methyl alcohol adds 12ml/LPTM, 2ml/L 0.02% vitamin H) and induces.Inducing culture 48-72 hour.The 50ml medium centrifugal is got supernatant, add ammonium sulfate to 20% saturation ratio, 4 ℃ of placements are spent the night behind the mixing, and the centrifuging and taking precipitation is with 3ml25mmol/LTris-HCl (pH7.0) dissolving, with Supdex 200 (Amersham Pharmacia Biotech UK) Φ 1.6 * 100cm column chromatography for separation, moving phase is 25mmol/LTris-HCl (pH7.0), and 150mmol/LNaCl, flow velocity are 1ml/L, ultraviolet detection, collect first peak (retention time is 70-85 minute), be the fusion rotein of purifying, SDS-PAGE analyzes and is the homogeneous band, as shown in Figure 5, wherein 1 is molecular weight standard, is respectively 94,67 from top to bottom, 43,30KD; 2 is the HSA/IL-2 sample before the purifying; 3-5 is the HSA/IL-2 of purifying.Activity (" Chinese biological goods rules " 2000 editions (the Chinese biological standard of articles council compiles, the 2000.6P396-398 of Chemical Industry Press) with CTLL-2 raji cell assay Raji HSA/IL-2.With the CTLL-2 cell cultures in 1640 substratum that contain 10% calf serum and 400-800IU/ml rhIL-2 (Gibco BRL USA).Go down to posterity weekly 3 times, 1640 substratum that contain 10% calf serum with preceding usefulness are washed cell 4 times, are diluted to 5 * 10
5Cell/ml.On 96 orifice plates, sample HSA/IL-2 does to do 2 times of dilutions after the suitably dilution on 96 orifice plates with 1640 substratum that contain 10% calf serum, every hole 50 μ l, and every hole adds cell 50 μ l, 5%CO again
2Cultivate after 18-24 hour for 37 ℃, add MTT solution (tetrazolium bromide solution, be made into the solution of 5.0mg/ml with phosphate buffered saline buffer, through 0.22 μ m membrane filtration degerming) 20 μ l, continue to cultivate 4-6 hour, add 20%SDS (sodium laurylsulfonate) solution, hatched 18-24 hour for 37 ℃, measure the absorbance of each sample well with enzyme connection instrument, with standard control, the activity of calculation sample HSA/IL-2, as shown in Figure 7, transverse axis is the hole count of 2 times of gradient dilutions, the longitudinal axis is an absorbance, and the activity that records purification of samples HSA/IL-2 is 3 * 10
6U/mg.Wrap by elisa plate with sample HSA/IL-2 dilution back, anti-with rabbit AHS albumin antibody respectively as one, the goat anti-rabbit igg of horseradish peroxidase-labeled is anti-as two, test sample HSA/IL-2, the result as shown in Figure 6, result from figure as can be known, sample HSA/IL-2 has the antigenicity of HSA.
Get the fusion rotein HSA/IL-2 and the rhIL-2 of identical active dose, the mouse tail vein administration, different time is got serum CTLL-2 cell (method is the same) mensuration IL-2 activity wherein.Compare with rhIL-2, the transformation period of fusion rotein HSA/IL-2 in mice plasma is about about 15h.
Sequence table
<160>4
<210>1
<211>585
<212>PRT
＜213〉Genus Homo people (Homo sapiens)
<400>1
Asp?Ala?His?Lys?Ser?Glu?Val?Ala?His?Arg?Phe?Lys?Asp?Leu?Gly
1 5 10 15
Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu?Ile?Ala?Phe?Ala?Gln?Tyr
20 25 30
Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val?Lys?Leu?Val?Asn?Glu
35 40 45
Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp?Glu?Ser?Ala?Glu
50 55 60
Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp?Lys?Leu?Cys
65 70 75
Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?MET?Ala?Asp?Cys
80 85 90
Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln?His
95 100 105
Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
110 115 120
Asp?Val?MET?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu
125 130 135
Lys?Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr
140 145 150
Ala?Pro?Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe
155 160 165
Thr?Glu?Cys?Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro
170 175 180
Lys?Leu?Asp?Glu?Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys
185 190 195
Gln?Arg?Leu?Lys?Cys?Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala
200 205 210
Phe?Lys?Ala?Trp?Ala?Val?Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys
215 220 225
Ala?Glu?Phe?Ala?Glu?Val?Ser?Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys
230 235 240
Val?His?Thr?Glu?Cys?Cys?His?Gly?Asp?Leu?Leu?Glu?Cys?Ala?Asp
245 250 255
Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile?Cys?Glu?Asn?Gln?Asp?Ser
260 265 270
Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu?Lys?Pro?Leu?Leu?Glu
275 280 285
Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp?Glu?MET?Pro?Ala
290 295 300
Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser?Lys?Asp?Val
305 310 315
Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly?Met?Phe
320 320 330
Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val?Leu
335 340 345
Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
350 355 360
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp
365 370 375
Glu?Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln
380 385 390
Asn?Cys?Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn
395 400 405
Ala?Leu?Leu?Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr
410 415 420
Pro?Thr?Leu?Val?Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser
425 430 435
Lys?Cys?Cys?Lys?His?Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu
440 445 450
Asp?Tyr?Leu?Ser?Val?Val?Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu
455 460 465
Lys?Thr?Pro?Val?Ser?Asp?Arg?Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser
470 475 480
Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe?Ser?Ala?Leu?Glu?Val?Asp?Glu
485 490 495
Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala?Glu?Thr?Phe?Thr?Phe?His
500 505 510
Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu?Arg?Gln?Ile?Lys?Lys
515 520 525
Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys?Pro?Lys?Ala?Thr
530 535 540
Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala?Ala?Phe?Val
545 550 555
Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe?Ala?Glu
560 565 570
Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly?Leu
575 580 585
<210>2
<211>133
<212>PRT
＜213〉Genus Homo people (Homo sapiens)
<400>2
Ala?Pro?Thr?Ser?Ser?Ser?Thr?Lys?Lys?Thr?Gln?Leu?Gln?Leu?Glu
1 5 10 15
His?Leu?Leu?Leu?Asp?Leu?Gln?Met?Ile?Leu?Asn?Gly?Ile?Asn?Asn
20 25 30
Tyr?Lys?Asn?Pro?Lys?Leu?Thr?Arg?Met?Leu?Thr?Phe?Lys?Phe?Tyr
35 40 45
Met?Pro?Lys?Lys?Ala?Thr?Glu?Leu?Lys?His?Leu?Gln?Cys?Leu?Glu
50 55 60
Glu?Glu?Leu?Lys?Pro?Leu?Glu?Glu?Val?Leu?Asn?Leu?Ala?Gln?Ser
65 70 75
Lys?Asn?Phe?His?Leu?Arg?Pro?Arg?Asp?Leu?Ile?Ser?Asn?Ile?Asn
80 85 90
Val?Ile?Val?Leu?Glu?Leu?Lys?Gly?Ser?Glu?Thr?Thr?Phe?Met?Cys
95 100 105
Glu?Tyr?Ala?Asp?Glu?Thr?Ala?Thr?Ile?Val?Glu?Phe?Leu?Asn?Arg
110 115 120
Trp?Ile?Thr?Phe?Cys?Gln?Ser?Ile?Ile?Ser?Thr?Leu?Thr
125 130 133
<210>3
<211>2253
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
ttcgaaacca?tgaagtgggt?aacctttatt?tcccttcttt?ttctctttag?ctcggcttat 60
tccaggggtg?tgtttcgtcg?agatgcacac?aagagtgagg?ttgctcatcg?gtttaaagat 120
ttgggagaag?aaaatttcaa?agccttggtg?ttgattgcct?ttgctcagta?tcttcagcag 180
tgtccatttg?aagatcatgt?aaaattagtg?aatgaagtaa?ctgaatttgc?aaaaacatgt 240
gtagctgatg?agtcagctga?aaattgtgac?aaatcacttc?ataccctttt?tggagacaaa 300
ttatgcacag?ttgcaactct?tcgtgaaacc?tatggtgaaa?tggctgactg?ctgtgcaaaa 360
caagaacctg?agagaaatga?atgcttcttg?caacacaaag?atgacaaccc?aaacctcccc 420
cgattggtga?gaccagaggt?tgatgtgatg?tgcactgctt?ttcatgacaa?tgaagagaca 480
tttttgaaaa?aatacttata?tgaaattgcc?agaagacatc?cttactttta?tgccccggaa 540
ctccttttct?ttgctaaaag?gtataaagct?gcttttacag?aatgttgcca?agctgctgat 600
aaagctgcct?gcctgttgcc?aaagctcgat?gaacttcggg?atgaagggaa?ggcttcgtct 660
gccaaacaga?gactcaaatg?tgccagtctc?caaaaatttg?gagaaagagc?tttcaaagca 720
tgggcagtgg?ctcgcctgag?ccagagattt?cccaaagctg?agtttgcaga?agtttccaag 780
ttagtgacag?atcttaccaa?agtccacacg?gaatgctgcc?atggagatct?gcttgaatgt 840
gctgatgaca?gggcggacct?tgccaagtat?atctgtgaaa?atcaggattc?gatctccagt 900
aaactgaagg?aatgctgtga?aaaacctctg?ttggaaaaat?cccactgcat?tgccgaagtg 960
gaaaatgatg?agatgcctgc?tgacttgcct?tcattagctg?ctgattttgt?tgaaagtaag 1020
gatgtttgca?aaaactatgc?tgaggcaaag?gatgtcttcc?tgggcatgtt?tttgtatgaa 1080
tatgcaagaa?ggcatcctga?ttactctgtc?gtgctgctgc?tgagacttgc?caagacatat 1140
gaaaccactc?tagagaagtg?ctgtgccgct?gcagatcctc?atgaatgcta?tgccaaagtg 1200
ttcgatgaat?ttaaacctct?tgtggaagag?cctcagaatt?taatcaaaca?aaattgtgag 1260
ctttttgagc?agcttggaga?gtacaaattc?cagaatgcgc?tattagttcg?ttacaccaag 1320
aaagtacccc?aagtgtcaac?tccaactctt?gtagaggtct?caagaaacct?aggaaaagtg 1380
ggcagcaaat?gttgtaaaca?tcctgaagca?aaaagaatgc?cctgtgcaga?agactatcta 1440
tccgtggtcc?tgaaccagtt?atgtgtgttg?catgagaaaa?cgccagtaag?tgacagagtc 1500
acaaaatgct?gcacagagtc?cttggtgaac?aggcgaccat?gcttttcagc?tctggaagtc 1560
gatgaaacat?acgttcccaa?agagtttaat?gctgaaacat?tcaccttcca?tgcagatata 1620
tgcacacttt?ctgagaagga?gagacaaatc?aagaaacaaa?ctgcacttgt?tgagcttgtg 1680
aaacacaagc?ccaaggcaac?aaaagagcaa?ctgaaagctg?ttatggatga?tttcgcagct 1740
tttgtagaga?agtgctgcaa?ggctgacgat?aaggagacct?gctttgccga?ggagggtaaa 1800
aaacttgttg?ctgcaagtca?agctgcctta?ggccttggtg?gtggtggatc?cgcacctact 1860
tcaagttcta?caaagaaaac?acagctacaa?ctggagcatt?tactgctgga?tttacagatg 1920
attttgaatg?gaattaataa?ttacaagaat?cccaaactca?ccaggatgct?cacatttaag 1980
ttttacatgc?ccaagaaggc?cacagaactg?aaacatcttc?agtgtctaga?agaagaactc 2040
aaacctctgg?aggaagtgct?aaatttagct?caaagcaaaa?actttcactt?aagacccagg 2100
gacttaatca?gcaatatcaa?cgtaatagtt?ctggaactaa?agggacctga?aacaacattc 2160
atgtgtgaat?atgctgatga?gacagcaacc?attgtagaat?ttctgaacag?atggattacc 2220
ttttgtcaaa?gcatcatctc?aacactgact?taa 2253
<210>4
<211>723
<212>PRT
＜213〉artificial sequence
<220>
<223>
<400>4
Asp?Ala?His?Lys?Ser?Glu?Val?Ala?His?Arg?Phe?Lys?Asp?Leu?Gly
1 5 10 15
Glu?Glu?Asn?Phe?Lys?Ala?Leu?Val?Leu?Ile?Ala?Phe?Ala?Gln?Tyr
20 25 30
Leu?Gln?Gln?Cys?Pro?Phe?Glu?Asp?His?Val?Lys?Leu?Val?Asn?Glu
35 40 45
Val?Thr?Glu?Phe?Ala?Lys?Thr?Cys?Val?Ala?Asp?Glu?Ser?Ala?Glu
50 55 60
Asn?Cys?Asp?Lys?Ser?Leu?His?Thr?Leu?Phe?Gly?Asp?Lys?Leu?Cys
65 70 75
Thr?Val?Ala?Thr?Leu?Arg?Glu?Thr?Tyr?Gly?Glu?MET?Ala?Asp?Cys
80 85 90
Cys?Ala?Lys?Gln?Glu?Pro?Glu?Arg?Asn?Glu?Cys?Phe?Leu?Gln?His
95 100 105
Lys?Asp?Asp?Asn?Pro?Asn?Leu?Pro?Arg?Leu?Val?Arg?Pro?Glu?Val
110 115 120
Asp?Val?MET?Cys?Thr?Ala?Phe?His?Asp?Asn?Glu?Glu?Thr?Phe?Leu
125 130 135
Lys?Lys?Tyr?Leu?Tyr?Glu?Ile?Ala?Arg?Arg?His?Pro?Tyr?Phe?Tyr
140 145 150
Ala?Pro?Glu?Leu?Leu?Phe?Phe?Ala?Lys?Arg?Tyr?Lys?Ala?Ala?Phe
155 160 165
Thr?Glu?Cys?Cys?Gln?Ala?Ala?Asp?Lys?Ala?Ala?Cys?Leu?Leu?Pro
170 175 180
Lys?Leu?Asp?Glu?Leu?Arg?Asp?Glu?Gly?Lys?Ala?Ser?Ser?Ala?Lys
185 190 195
Gln?Arg?Leu?Lys?Cys?Ala?Ser?Leu?Gln?Lys?Phe?Gly?Glu?Arg?Ala
200 205 210
Phe?Lys?Ala?Trp?Ala?Val?Ala?Arg?Leu?Ser?Gln?Arg?Phe?Pro?Lys
215 220 225
Ala?Glu?Phe?Ala?Glu?Val?Ser?Lys?Leu?Val?Thr?Asp?Leu?Thr?Lys
230 235 240
Val?His?Thr?Glu?Cys?Cys?His?Gly?Asp?Leu?Leu?Glu?Cys?Ala?Asp
245 250 255
Asp?Arg?Ala?Asp?Leu?Ala?Lys?Tyr?Ile?Cys?Glu?Asn?Gln?Asp?Ser
260 265 270
Ile?Ser?Ser?Lys?Leu?Lys?Glu?Cys?Cys?Glu?Lys?Pro?Leu?Leu?Glu
275 280 285
Lys?Ser?His?Cys?Ile?Ala?Glu?Val?Glu?Asn?Asp?Glu?MET?Pro?Ala
290 295 300
Asp?Leu?Pro?Ser?Leu?Ala?Ala?Asp?Phe?Val?Glu?Ser?Lys?Asp?Val
305 310 315
Cys?Lys?Asn?Tyr?Ala?Glu?Ala?Lys?Asp?Val?Phe?Leu?Gly?Met?Phe
320 320 330
Leu?Tyr?Glu?Tyr?Ala?Arg?Arg?His?Pro?Asp?Tyr?Ser?Val?Val?Leu
335 340 345
Leu?Leu?Arg?Leu?Ala?Lys?Thr?Tyr?Glu?Thr?Thr?Leu?Glu?Lys?Cys
350 355 360
Cys?Ala?Ala?Ala?Asp?Pro?His?Glu?Cys?Tyr?Ala?Lys?Val?Phe?Asp
365 370 375
Glu?Phe?Lys?Pro?Leu?Val?Glu?Glu?Pro?Gln?Asn?Leu?Ile?Lys?Gln
380 385 390
Asn?Cys?Glu?Leu?Phe?Glu?Gln?Leu?Gly?Glu?Tyr?Lys?Phe?Gln?Asn
395 400 405
Ala?Leu?Leu?Val?Arg?Tyr?Thr?Lys?Lys?Val?Pro?Gln?Val?Ser?Thr
410 415 420
Pro?Thr?Leu?Val?Glu?Val?Ser?Arg?Asn?Leu?Gly?Lys?Val?Gly?Ser
425 430 435
Lys?Cys?Cys?Lys?His?Pro?Glu?Ala?Lys?Arg?Met?Pro?Cys?Ala?Glu
440 445 450
Asp?Tyr?Leu?Ser?Val?Val?Leu?Asn?Gln?Leu?Cys?Val?Leu?His?Glu
455 460 465
Lys?Thr?Pro?Val?Ser?Asp?Arg?Val?Thr?Lys?Cys?Cys?Thr?Glu?Ser
470 475 480
Leu?Val?Asn?Arg?Arg?Pro?Cys?Phe?Ser?Ala?Leu?Glu?Val?Asp?Glu
485 490 495
Thr?Tyr?Val?Pro?Lys?Glu?Phe?Asn?Ala?Glu?Thr?Phe?Thr?Phe?His
500 505 510
Ala?Asp?Ile?Cys?Thr?Leu?Ser?Glu?Lys?Glu?Arg?Gln?Ile?Lys?Lys
515 520 525
Gln?Thr?Ala?Leu?Val?Glu?Leu?Val?Lys?His?Lys?Pro?Lys?Ala?Thr
530 535 540
Lys?Glu?Gln?Leu?Lys?Ala?Val?Met?Asp?Asp?Phe?Ala?Ala?Phe?Val
545 550 555
Glu?Lys?Cys?Cys?Lys?Ala?Asp?Asp?Lys?Glu?Thr?Cys?Phe?Ala?Glu
560 565 570
Glu?Gly?Lys?Lys?Leu?Val?Ala?Ala?Ser?Gln?Ala?Ala?Leu?Gly?Leu
575 580 585
Gly?Gly?Gly?Gly?Ser?Ala?Pro?Thr?Ser?Ser?Ser?Thr?Lys?Lys?Thr
590 595 600
Gln?Leu?Gln?Leu?Glu?His?Leu?Leu?Leu?Asp?Leu?Gln?Met?Ile?Leu
605 610 615
Asn?Gly?Ile?Asn?Asn?Tyr?Lys?Asn?Pro?Lys?Leu?Thr?Arg?Met?Leu
620 625 630
Thr?Phe?Lys?Phe?Tyr?Met?Pro?Lys?Lys?Ala?Thr?Glu?Leu?Lys?His
635 640 645
Leu?Gln?Cys?Leu?Glu?Glu?Glu?Leu?Lys?Pro?Leu?Glu?Glu?Val?Leu
650 655 660
Asn?Leu?Ala?Gln?Ser?Lys?Asn?Phe?His?Leu?Arg?Pro?Arg?Asp?Leu
665 670 675
Ile?Ser?Asn?Ile?Asn?Val?Ile?Val?Leu?Glu?Leu?Lys?Gly?Ser?Glu
680 685 690
Thr?Thr?Phe?Met?Cys?Glu?Tyr?Ala?Asp?Glu?Thr?Ala?Thr?Ile?Val
695 700 705
Glu?Phe?Leu?Asn?Arg?Trp?Ile?Thr?Phe?Cys?Gln?Ser?Ile?Ile?Ser
710 715 720
Thr?Leu?Thr
723
Claims (10)
1, the fusion rotein of a kind of human serum albumin and interleukin II, comprise with sequence table in SEQ ID №: 1 have at least 85% sequence homology amino acid residue sequence first district and with sequence table in SEQ ID №: 2 have second district of the amino acid residue sequence of at least 85% sequence homology.
2, fusion rotein according to claim 1, it is characterized in that: the fusion rotein of described human serum albumin and interleukin II comprises SEQ ID № in the sequence table: SEQ ID № in first district of 1 amino acid residue sequence and the sequence table: second district of 2 amino acid residue sequence, or the function equivalent in above-mentioned two districts; Described function equivalent is under the prerequisite that does not change described fusion rotein characteristic, the polypeptide that replacement, disappearance or the interpolation of the indivedual amino-acid residues of described fusion rotein obtained.
3, fusion rotein according to claim 2 is characterized in that: described replacement occurs in SEQ ID №: 2 from N-terminal the 125th halfcystine.
4, fusion rotein according to claim 3 is characterized in that: described the 125th halfcystine replaced by Serine or L-Ala.
5, according to claim 1,2,3 or 4 described fusion roteins, it is characterized in that: SEQ ID № in the described and sequence table: SEQ ID № in 1 homologous, first district and the sequence table: 2 homologous are provided with connection peptides between second district, and the general formula of described connection peptides is [GlyGlyGlyGlySer]
n, n is the integer of 1-10, is preferably the integer of 1-3.
6, fusion rotein according to claim 5 is characterized in that: the fusion egg of described human serum albumin and interleukin II is for having SEQ ID № in the sequence table: the protein of 4 amino acid residue sequences.
7, the dna sequence dna of the described fusion rotein of arbitrary claim among the coding claim 1-6.
8, dna sequence dna according to claim 7 is characterized in that: described dna sequence dna is SEQ ID №: 3.
9, the expression vector and the clone that contain the described dna sequence dna of claim 7.
10, the application of the fusion rotein of described human serum albumin of claim 1 and interleukin II in the medicine of preparation human interleukin-12 class medicine and/or preparation treatment and human interleukin-12 or its acceptor overexpression diseases related.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB200310117068XA CN100379762C (en) | 2003-12-08 | 2003-12-08 | Interfusion protein between human serum albumin and interleukin, and encoding genes |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200310117068XA CN100379762C (en) | 2003-12-08 | 2003-12-08 | Interfusion protein between human serum albumin and interleukin, and encoding genes |
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| Publication Number | Publication Date |
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| CN1626554A true CN1626554A (en) | 2005-06-15 |
| CN100379762C CN100379762C (en) | 2008-04-09 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB200310117068XA Expired - Lifetime CN100379762C (en) | 2003-12-08 | 2003-12-08 | Interfusion protein between human serum albumin and interleukin, and encoding genes |
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| CN (1) | CN100379762C (en) |
Cited By (8)
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| CN1313494C (en) * | 2005-08-29 | 2007-05-02 | 中国人民解放军军事医学科学院生物工程研究所 | Fusion protein possessing growth facilitation action and its coding gene and uses |
| CN100371349C (en) * | 2006-04-13 | 2008-02-27 | 广州医学院第二附属医院 | Recombinant immunotherapeutic protein and its expression method and application |
| EA014422B1 (en) * | 2008-01-25 | 2010-12-30 | Михаил Николаевич Смирнов | PICHIA PASTORIS YEAST STRAIN PS107(pPIC9HAbIL-2), WHICH PRODUCES A HYBRID PROTEIN CONSISTING OF HUMAN BLOOD PLASMA ALBUMIN AND HUMAN INTERLEUKIN-2, RECOMBINANT PLASMID pPIC9HAbIL-2 AND A METHOD OF CONSTRUCTION THEREOF |
| CN105198999A (en) * | 2014-05-27 | 2015-12-30 | 上海生物制品研究所有限责任公司 | Fusion protein and its preparation method and use |
| CN109689694A (en) * | 2016-05-19 | 2019-04-26 | 通用医疗公司 | Interleukin 2 in conjunction with its receptor IL-2R β, which is used as, to be used to enhance natural killer cells and the active platform of regulatory T cells |
| CN109762069A (en) * | 2018-11-05 | 2019-05-17 | 广州安辰新药研究院有限公司 | A kind of fusion protein, its pharmaceutical composition and purposes |
| CN110950967A (en) * | 2019-12-13 | 2020-04-03 | 山东民康生物科技有限公司 | Anti-human serum albumin nano antibody and IL-2 fusion protein and preparation method thereof |
| WO2023024759A1 (en) * | 2021-08-25 | 2023-03-02 | 北京伟德杰生物科技有限公司 | Fusion protein of interleukin-2 and application thereof in als |
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| CN112724259B (en) * | 2020-11-16 | 2022-12-20 | 天津林达生物科技有限公司 | Fusion protein of human serum albumin and interleukin 2 and application thereof |
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| CA2747325A1 (en) * | 2000-04-12 | 2001-10-25 | Human Genome Sciences, Inc. | Albumin fusion proteins |
| CN1405183A (en) * | 2001-08-10 | 2003-03-26 | 中国人民解放军军事医学科学院生物工程研究所 | Serum albumin and granulocyte-macrophage colony stimulating factor fusion protein |
| CN101200503B (en) * | 2001-08-10 | 2010-07-07 | 中国人民解放军军事医学科学院生物工程研究所 | Fusion protein for seralbumin and interferon |
| CN1405182A (en) * | 2001-08-10 | 2003-03-26 | 中国人民解放军军事医学科学院生物工程研究所 | Serum albumin and granulocyte colony stimulating factor fusion protein |
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2003
- 2003-12-08 CN CNB200310117068XA patent/CN100379762C/en not_active Expired - Lifetime
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| CN1313494C (en) * | 2005-08-29 | 2007-05-02 | 中国人民解放军军事医学科学院生物工程研究所 | Fusion protein possessing growth facilitation action and its coding gene and uses |
| CN100371349C (en) * | 2006-04-13 | 2008-02-27 | 广州医学院第二附属医院 | Recombinant immunotherapeutic protein and its expression method and application |
| EA014422B1 (en) * | 2008-01-25 | 2010-12-30 | Михаил Николаевич Смирнов | PICHIA PASTORIS YEAST STRAIN PS107(pPIC9HAbIL-2), WHICH PRODUCES A HYBRID PROTEIN CONSISTING OF HUMAN BLOOD PLASMA ALBUMIN AND HUMAN INTERLEUKIN-2, RECOMBINANT PLASMID pPIC9HAbIL-2 AND A METHOD OF CONSTRUCTION THEREOF |
| CN105198999A (en) * | 2014-05-27 | 2015-12-30 | 上海生物制品研究所有限责任公司 | Fusion protein and its preparation method and use |
| US12202856B2 (en) | 2016-05-19 | 2025-01-21 | The General Hospital Corporation | Tethered interleukin-2 to its receptor IL-2RBETA, a platform to enhance natural killer and regulatory T cell activity |
| CN109689694A (en) * | 2016-05-19 | 2019-04-26 | 通用医疗公司 | Interleukin 2 in conjunction with its receptor IL-2R β, which is used as, to be used to enhance natural killer cells and the active platform of regulatory T cells |
| CN109762069A (en) * | 2018-11-05 | 2019-05-17 | 广州安辰新药研究院有限公司 | A kind of fusion protein, its pharmaceutical composition and purposes |
| CN109762069B (en) * | 2018-11-05 | 2022-01-14 | 广州领晟医疗科技有限公司 | Fusion protein, pharmaceutical composition and application thereof |
| CN110950967A (en) * | 2019-12-13 | 2020-04-03 | 山东民康生物科技有限公司 | Anti-human serum albumin nano antibody and IL-2 fusion protein and preparation method thereof |
| CN110950967B (en) * | 2019-12-13 | 2022-05-13 | 山东民康生物科技有限公司 | Anti-human serum albumin nano antibody and IL-2 fusion protein and preparation method thereof |
| WO2023024759A1 (en) * | 2021-08-25 | 2023-03-02 | 北京伟德杰生物科技有限公司 | Fusion protein of interleukin-2 and application thereof in als |
| WO2023024215A1 (en) * | 2021-08-25 | 2023-03-02 | 北京伟德杰生物科技有限公司 | Long-acting recombinant human interleukin-2 fusion protein, and preparation method therefor and application thereof |
| WO2023024758A1 (en) * | 2021-08-25 | 2023-03-02 | 北京伟德杰生物科技有限公司 | Fusion protein of interleukin 2 and application thereof in ibd |
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