The application be submit to August 10 calendar year 2001, application number is 01124110.1, denomination of invention is divided an application for the Chinese patent application of " fusion rotein of serum albumin and Interferon, rabbit ".
Summary of the invention
Serum albumin and interferon fusion protein that to the purpose of this invention is to provide a kind of characteristic that collects serum albumin and Interferon, rabbit be one.
The fusion rotein of serum albumin of the present invention and Interferon, rabbit, comprise with first district of human serum albumin at least 85% sequence homology and with second district of human interferon at least 85% sequence homology.
Wherein preferably comprise with first district of human serum albumin at least 95% sequence homology and with second district of human interferon at least 95% sequence homology.
Most preferably comprise first district identical and second district identical with the human interferon amino acid residue sequence with the human serum albumin 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 adding of the indivedual amino-acid residues of fusion rotein obtained.
In fusion rotein of the present invention, can be the N-end that is positioned at fusion rotein with human serum albumin homologous first district, be positioned at the C-end of fusion rotein with human interferon homologous second district; Also can be the N-end that is positioned at fusion rotein with human interferon homologous second district, be positioned at the C-end of fusion rotein with human serum albumin homologous first district, but the expression of the gene of the previous case in yeast be higher.
In order to make bigger interval is arranged between fusion rotein two portions, the Interferon, rabbit part is combined with Interferon Receptors most possibly, be provided with connection peptides between described and human serum albumin homologous first district and human interferon homologous second district, the general formula of described connection peptides is [GlyGly GlyGly Ser]
n, n is the integer of 1-10; Wherein, preferably n is the integer of 1-3.
Interferon, rabbit of the present invention can be IFN α, IFN β, also can be IFN γ, when Interferon, rabbit is IFN α, can be IFN α 2a, IFN α 1b, IFN α 2b or total Interferon, rabbit IFN α con.
Another object of the present invention provides the dna sequence dna of code book invention fusion rotein.
The dna sequence dna that comprises the fusion rotein in first district identical and second district identical with the human interferon amino acid residue sequence with the human serum albumin amino acid residue sequence.Wherein first district is identical with the human serum albumin amino acid residue sequence, and second district is identical with the human interferon amino acid residue sequence, and the centre is provided with the fusion rotein of GlyGlyGlyGlySer connection peptides.
Another purpose of the present invention provides the recombinant expression vector of the dna sequence dna that carries code book invention fusion rotein.
Recombinant expression vector of the present invention comprises pHIL-D2HSAIFN plasmid, PD3HSAIFN0 plasmid, PD3HSAIFN2 plasmid, pPIC9IFN-HSA plasmid etc.
A further object of the present invention provides the host who expresses fusion rotein encoding gene of the present invention.
Host of the present invention can be transformed by the part of recombinant expression vector or recombinant expression vector, contains bacterium, yeast, zooblast or the vegetable cell of fusion rotein encoding gene of the present invention; Wherein preferably yeast, more preferably pichia spp, most preferably pichia spp GS115.
The present invention is with the albumin molecule, or the analogue of its at least 85% sequence homology and Interferon, rabbit, or the fusion of the analogue of its at least 85% sequence homology, formed fusion rotein had both kept and Interferon Receptors bonded activity, simultaneously compare the prolongation of having got back its plasma half-life with Interferon, rabbit.Fusion rotein of the present invention both can keep the activity that activates Interferon Receptors, agonist as this receptor, manufacture the interferons medicine, also can only keep and this receptor bonded activity, as the inhibitor of this receptor, manufacture the medicine that is used for the treatment of some and Interferon, rabbit or its acceptor overexpression diseases related.
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 hIFN 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.The polynucleotide of coding IFN α (comprising IFN α 1b, IFN α 2b, IFN α 2a etc.) can obtain from the RNA goods with new castle disease virus inductive human peripheral leucocytes with the method for RT-PCR, the polynucleotide of coding IFN α con can obtain with artificial synthetic method, and the polynucleotide of coding IFN β can obtain from the RNA goods with Interferon, rabbit, Polyinosinic-polycytidylic acid, cycloheximide and actinomycin inductive human fibroblasts, amnion cell, skin cancer cell with the method for RT-PCR.If needing available method well known in the art suddenlys change, lacks, inserts and is connected with other polynucleotide polynucleotide etc.The fusion of the polynucleotide of coding HSA and coding hIFN, 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 hIFN, thereby obtains the gene of encoding fusion protein.Can introduce polynucleotide in the both sides of encoding fusion protein gene of the present invention if desired, 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 the narration of (J. Sa nurse Brooker etc., " molecular cloning experiment guide " second edition, Science Press, 1995.) such as J. Sa nurse Brookers.
Many expression vectors 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 fusion rotein in the code book 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 of expressed fusion protein can be yeast, mammalian cell, bacterium, animal, plant etc.Fusion rotein or polypeptide may reside in the host cell, also can be that secretion is come out from the host, and be preferred, 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.
Transform required nucleic acid and to host cell, remove available usual method, as: electroporation prepares competent spheroplast etc.The success cell transformed promptly contains the cell of DNA construct 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.Perhaps, in the cells and supernatant or the albumen in the cytoclasis liquid can be with the antibody test of anti-HSA or anti-IFN.
Can contain the host of DNA construct 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 with shaking bottle or bio-reactor etc., bio-reactor preferably during production.Substratum should be able to provide thalline (or cell) growth and product to express required material, 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 divide 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 in the cell culture that contain DNA construct of the present invention, separate with the method for various albumen sepn, purified fusion protein.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 among 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 on the amino of polypeptide, carboxyl, hydroxyl, sulfydryl, modifying again.Used modifier can but be not limited to polyoxyethylene glycol, dextran etc.
Fusion rotein among 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, and can prepare by any methods known in the art.These methods comprise fusion rotein and have 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.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.Freeze-dried preparation adds aseptic, pyrogen-free liquid carrier before using, as water for injection with liquid preparation lyophilize preparation.
Preferred dosage unit includes the daily dosage of daily dosage or unit or its suitable Asia branch of fusion rotein.
Fusion rotein among 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.Treatment is included in and uses single dose or compound dosage in for some time.
Fusion rotein of the present invention and derivative thereof or its pharmaceutical composition, can be used for treating the disease of plain or its analogue treatment of available interference as the interferons medicine, as viral hepatitis (comprise B-mode, hepatitis C etc.), tumour (comprising leukemia, some solid tumors etc.), multiple sclerosis, similar rheumatism etc.Can be used for treatment and Interferon, rabbit or its acceptor overexpression diseases associated as the Interferon, rabbit inhibitor, as transformation reactions etc.
Using dosage can calculate with respect to tiring of IFN by fusion rotein, considers the transformation period that fusion rotein prolongs with respect to natural IFN simultaneously.Typical amounts is 1 * 10
3-1 * 10
6U/kg.In by total length HSA and total length IFN fusion rotein, mean at once mutually according to mole number and to use the greater weight fusion rotein, but frequency of utilization can reduce, as weekly or still less.
The present invention will be further described below in conjunction with specific embodiment.
Embodiment
The clone of embodiment 1: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, wherein primer HSA2 has also added the sequence of coding GlyGlyGlyGlySer joint peptide behind its 3 ' end except that the 3 ' end sequence that comprises the HSA gene.
HSA1:5’GC
TTCGAAACCATGAAGTGGGTAACCTTTATTTCCCT3’
HSA2:5’TA
GGATCCACCACCACCAAGGCCTAAGGCAGCTTGACTTGC3’
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 20 μ mol/L and HSA2 primer, the dNTP of 2mmol/L, 10 μ l, 10X 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 carrying of μ l pGEM-T, 8 μ l 2X damping fluids, 1 μ lT4DNA 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 is assisted on the LB agar plate that contains x-gal and IPTG and penbritin (50 μ g/ml), 37 ℃ of overnight incubation.The picking white colony is seeded to the LB substratum that 3ml contains penbritin (50 μ g/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 the clone of the correct HSAcDNA of sequence, JM109 (pGEMT-HSA), here Ke Long HSA cDNA contains the encoding sequence of its natural signals peptide and preceding peptide moiety, so that when expressing, directly utilize its signal peptide and propetide to be used for, added the encoding sequence of joint peptide GlyGlyGlyGlySer simultaneously at the c of HSA cDNA end as secretion signal.
The cDNA clone of embodiment 2:IFN α
Get normal people's peripheral blood and add and separate lymphocyte layering liquid, the separation of human white corpuscle with the DMEM culture medium culturing that contains 5% foetal calf serum, adds behind the new castle disease virus 37 ℃ and cultivated 1 hour, and the centrifugal supernatant of abandoning adds substratum again, and 37 ℃, 5%CO
2Cultivated 18 hours.Extract test kit with RNA and separate total RNA, (these two test kits are all available from vast Tyke, Beijing biological gene technology company limited with general RT-PCR test kit, the concrete operations by specification carries out) obtain the cDNA of reverse transcription, obtain the cDNA of IFN α by PCR method.Used primer is slightly different with the difference of required clone's IFN alpha hypotype, and as clone IFN α 2b, IFN α 2a, its primer can be used:
IFNα2b-1:5’---ATGGATCC?TGT?GAT?CTC?CCT?CAA?ACC?CAC?AG---3’
IFNα2b-2:5’---ATGAATTC?TTA?TTC?CTT?ACT?CCT?TAA?TCT?TTC?TTG---3’
Clone IFN α 1b, its primer can be used:
IFNα?1b-1:5’---ATGGATCC?TGT?GAT?CTC?CCT?GAG?ACC?CAC?AG---3’
IFNα?1b-2:5’---ATGAATTC?TTA?TTC?CTT?CCT?CCT?TAA?TCT?TTC?TTG---3’
PCR method is:
Add 1 μ l reverse transcription product in the 100 μ l reaction systems, each 3 μ l of the IFN α 2b-1 of 20 μ mol/L, IFN α 2b-2 (or IFN α 1b-1, IFN α 1b-2) primer, the dNTP of 2mmol/L, 10 μ l, 10X 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 1.5 minutes, and circulated 35 times.Show the band of an expection big or small (about 0.5kb) 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 (the pfu enzyme in the experiment on the pUC19 plasmid of BamHI/EcoRI enzymolysis, restriction endonuclease, ligase enzyme, the pUC19 plasmid, test kits etc. are available from vast Tyke biological gene technology company limited of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and Beijing), transformed into escherichia coli JM109, every kind of PCR product is selected some clones, plasmid purification, BamHI-EcoRI district dna sequencing, select its amino acid sequence coded and contain IFN α 1b respectively, IFN α 2b, the clone of IFN α 2a sequence, called after pUC-IFN α 1b respectively, pUC-IFN α 2b, pUC-IFN α 2a etc.The cDNA of the IFN α of other hypotype can clone with similar methods, and the gene of total Interferon, rabbit can obtain with the method for synthetic.
The cDNA clone of embodiment 3:IFN β
Human amniotic cell (Wish cell, Chinese Academy of Sciences's cell bank provides) be paved with bottle with 1640 culture medium culturing to the cell that contains 10% calf serum at the bottom of, abandon substratum, add and contain 100U/ml IFN, continue to cultivate 16 hours with 1640 substratum of 10% calf serum, abandon substratum.Add 1640 substratum that contain 50 μ g/ml polyinosinic acids and 10 μ g/ml cycloheximides and continue to cultivate 4 hours, add unwrapping wire rhzomorph D1 μ g/ml again, cultivated 5 hours.Abandon supernatant, after trysinization, collecting cell, extract test kit with RNA and separate total RNA, (these two test kits are all available from vast Tyke, Beijing biological gene technology company limited with general RT-PCR test kit, the concrete operations by specification carries out) obtain the cDNA of reverse transcription, obtain the cDNA of IFN β by the method for PCR, its primer can be used:
IFNβ-1:5’ATGGATCC?ATG?AGC?TAC?AAC?TTG?CTT?GGA3’
IFNβ-2:5’ATGAATTC?TTA?GTT?TCG?GAG?GTA?ACC?TGT?AAG?TC3’
PCR and cloning process be with embodiment 2, and the clone of acquisition BamHI-EcoRI district dna sequencing selects the clone of its amino acid sequence coded and IFN β sequence, called after pUC-IFN β.
Embodiment 4: Expression of Fusion Protein of the present invention when connection peptides is GlyGlyGlyGly Ser
As shown in Figure 1, for HSA and IFN with the fusion rotein form from the pichia spp secreting, expressing, select the pHIL-D2 plasmid as carrier (Invitrogen Corp.USA).Between this carrier A OX promotor downstream NspV and EcoRI site, insert the HSA/IFN 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/Sal enzyme, reclaim the 4kb fragment, self connect back called after pD3 carrier.The pD3 carrier is cut with NspV/EcoRI, reclaim linearized vector, with embodiment 2, the 3 pUC19-IFN α 1b that make up, pUC19-IFN α 2b, pUC19-IFN α 2a and pUC19-IFN β cut with BamHI/EcoRI respectively, reclaim the fragment of the cDNA that contains IFN of about 0.5kb, the pGEMT-HSA that embodiment 1 is made up cuts with NspV/BamHI, reclaim the fragment that contains HSA cDNA of about 1.8kb, with linearizing pD3 carrier with contain HSAcDNA and be connected with the catalysis of T4 ligase enzyme respectively with the fragment that respectively contains IFN cDNA, transformed into escherichia coli JM109, select positive colony, cut evaluation with NspV/EcoRI and NspV/BamHI enzyme, obtain positive colony e. coli jm109 (pD3HSAIFN α 1b), e. coli jm109 (pD3HSAIFN α 2b), e. coli jm109 (pD3HSA IFN α 2a) and e. coli jm109 (pD3HSA IFN β).After above-mentioned clone cultivated amplification respectively, plasmid purification was cut with ClaI/ScaI, and electrophoresis reclaims linearizing about 6.3kb fragment, and the pHIL-D2 empty carrier is cut with ClaI/ScaI, reclaimed the fragment of about 4.2kb.The 4.2kb fragment of pHIL-D2 is connected with the catalysis of T4 ligase enzyme with the 6.3kb fragment of above-mentioned HSA of containing and various IFN genes respectively, after connecting product transformed into escherichia coli JM109, assist in the LB agar plate that contains penbritin (50ug/ml), select positive colony, the extracting plasmid, enzyme is cut evaluation, obtain clone's e. coli jm109 (pHIL-D2HSAIFN α 1b), e. coli jm109 (pHIL-D2HSAIFN α 2b), e. coli jm109 (pHIL-D2HSAIFN α 2a) and e. coli jm109 (pHIL-D2HSAIFN β).The building process of these plasmids is (wherein IFN represents IFN α 1b, various and each hypotype Interferon, rabbit such as IFN α 2b, IFN β) as shown in Figure 1.After these clones cultivated amplification respectively, the about 10 μ g of each plasmid purification cut with the NotI enzyme.Transform pichia spp GS115 (the yeast expression test kit is provided by Invitrogen Corp.USA) after the linearizing respectively, the GS115 after the conversion assists respectively in the RDB agar that contains sorbyl alcohol [1.5% agar, 1mol/L sorbyl alcohol, 1% glucose, 4 * 10
-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%)] plate, cultivate 1-2 weeks, picking His for 37 ℃
+The clone.Be seeded to 25mlBMGY substratum (100mmol/L potassiumphosphate, pH6.0,4 * 10 are housed
-5The % 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 ℃.With cytopathic-effect inhibition assay measure Interferon, rabbit in the culture supernatant activity (method is seen " Chinese biological goods rules " 2000 editions, and the Chinese biological standard of articles council compiles, Chemical Industry Press 2000.6, p373-375).Human amniotic cell (Wish cell) is incubated in the MEM substratum that contains 10% calf serum, go down to posterity weekly and (use trysinization with preceding, be made into 2.5-3.5 * 10 with the MEM substratum that contains 10% calf serum for 2 times
5Individual cell/ml).Be inoculated in the 96 porocyte culture plates every hole 100 μ l, 37 ℃, 5%CO
2Cultivated 4-6 hour.In another 96 porocyte culture plate, every hole adds 150 μ l and measures nutrient solution (the MEM substratum that contains 7% calf serum), and sample is made 2 times of gradient dilutions, and every hole 100 μ l sample diluting liquids are transferred in the culture plate that is added with cell, and 37 ℃, 5%CO
2Cultivated 18-24 hour, and abandoned supernatant, add and use the MEM substratum that contains 3% calf serum to be diluted to 100TCID
50VSV virus (vesicular stomatitis virus), every hole 100 μ l, 37 ℃, 5%CO
2Cultivated 24 hours, abandon supernatant, every hole adds 50 μ l staining fluids (50mg Viola crystallina is dissolved in 20% ethanol), room temperature was placed 30 minutes, flowing water carefully washes away staining fluid, blots residual moisture, and every hole adds 100 μ l destainers (0.1% acetate, 50% ethanol), room temperature was placed 5 minutes, measured its absorbance at 570nm.At same extent of dilution, the clone that absorbancy is high promptly is a high-expression clone.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 Interferon, rabbit, and the activation analysis proof has the activity of interferon anti-reflecting virus.
Embodiment 5:IFN/HSA Expression of Fusion Protein
With the human peripheral leucocytes cDNA of embodiment 2 preparations, obtain the cDNA of IFN α by PCR method.Used primer is slightly different with the difference of the hypotype of required clone's IFN α, as clone IFN α 2b, and IFN α 2a, its primer can be used:
IFNα?2b-3:5’AT
GTCGAC?
CTCGAG?AAA?AGA?TGT?GAT?CTC?CCT?CAA?ACCCAC?AG3’
IFNα?2b-4:5’AT
GGATCCACC?ACC?GCC?TTC?C?TT?ACT?CCT?TAA?TCT?TTCTTG3’
Clone IFN α 1b, its primer can be used:
IFNα?1b-3:5’ATGTCGAC?CTCGAG?AAA?AGA?TGT?GAT?CTC?CCT?GAG?ACCCAC?AG3’
IFNα?1b-4:5’AT_GGATCCACC?ACC?GCC?TTC?CTT?CCT?CCT?TAA?TCT?TTCTTG3
The cDNA of the IFN α of other hypotype can clone with similar methods, and the gene of total Interferon, rabbit can obtain with the method for synthetic.
The cDNA that obtains with embodiment 3 reverse transcriptions is a template, obtains the cDNA of IFN β by PCR method.
Its primer can be used:
IFNβ-3:5’---ATGTCGAC?CTCGAG?AAA?AGA?ATG?AGC?TAC?AAC?TTG?CTTGGA3’
IFNβ-4:5’---ATGGATCCACC?ACC?GCC?GTT?TCG?GAG?GTA?ACC?TGT?AAGTC3’
Primer is synthetic with the oligonucleotide synthesizer.PCR method is with embodiment 1, behind the pcr amplification, introduce a Sal I site and an Xho I site at 5 ' of cDNA-end, KEX2 restriction enzyme site has simultaneously induced one in downstream, Xho I site, in order to excise the signal peptide of the fusion rotein of expressing, 3 '-end is introduced BamH I site.Behind the PCR product electrophoresis purifying, be cloned on pUC19 (vast Tyke, Beijing biological gene technology company limited) plasmid with Sal I/BamHI enzymolysis, form pUC-IFN α 2b2, pUC-IFN α 1b2, pUC-IFN β 2 etc.The dna sequencing result shows that these clones contain the sequence of coding IFN α 2b, IFN α 1b and IFN β respectively, just Sal I site, Xho I site, KEX2 restriction enzyme site have been added at its 5 '-end, removed terminator codon with 3 '-end, the oligonucleotide that has added the coding flexible joint, and BamHI site.
Method with PCR obtains HSA cDNA from people liver tire cDNA library (available from Clontech Laboratories Inc.USA), used primer HSA5 and HSA6 are synthetic with the oligonucleotide synthesizer:
HSA5:5’—TA
GGATCC?GAT?GCA?CAC?AAG?AGT?GAG?GTT—3’
HSA6:5’—ATGAATTCTTAAAGGCCTAAGGCAGCTTGACTTGC—3’
PCR method, is cloned on pUC19 (vast Tyke, Beijing biological gene technology company limited) plasmid with the BamHI/EcoRI enzymolysis behind the PCR product electrophoresis purifying with embodiment 1, forms pUCHSA2.The dna sequencing result shows that this cloned sequence is identical with the encoding sequence of HSA mature peptide, just adds the EcoRI site after its 5 '-end has added BamHI site and 3 '-end terminator codon.
Select the pPIC9 plasmid as Yeast expression carrier (Invitrogen Corp.USA).Between this carrier A OX promotor downstream XhoI and EcoRI site, insert the IFN/HSA gene.The pUC-IFN α 2b2, the plasmids such as pUC-IFN α 1b2, pUC-IFN β 2 that make up are above cut with XhoI/BamHI, reclaimed the fragment that contains IFN cDNA of 0.5kb respectively; PUCHSA2 is cut with BamHI/EcoR I, reclaim the fragment that contains HSA cDNA of about 1.8kb; The pPIC9 plasmid is cut with XhoI/EcoR I enzyme, reclaim the linearizing fragment, various IFN cDNA are connected with the catalysis of T4DNA ligase enzyme with linearizing pPIC9 carrier with the fragment that contains HSAcDNA respectively, transformed into escherichia coli JM109, select positive colony, cut evaluation with XhoI/BamHI and BamHI/EcoR I enzyme respectively, obtain positive colony e. coli jm109 (pPIC9IFN α 2b HSA), e. coli jm109 (pPIC9 IFN α 1b HSA), e. coli jm109 (pPIC9 IFN β HSA).Behind each clonal expansion, plasmid purification, behind the BglII linearization for enzyme restriction, transform pichia spp GS115 (method that Invitrogen Corp.USA company yeast expression test kit provides), GS115 after the conversion, assist in the RDB agar plate that contains sorbyl alcohol (prescription is with embodiment 3), cultivate 1-2 week, picking His for 37 ℃
+The clone.Shake-flask culture and expression strain screening method are with embodiment 3, and the cultivation of engineering zymic, product purification, IFN determination of activity, HSA antigenic determination method are with embodiment 7.The result shows the antigenicity that the IFN/HSA fusion rotein of expression has stimulates IFN activity and HSA.
Embodiment 6: different joint HSA/IFN Expression of Fusion Protein
With the plasmid pD3HSAIFN α 1b that makes up among the embodiment 4, pD3HSAIFN α 2b, pD3HSAIFN α 2a, pD3HSAIFN β etc. uses BamH I/Stu I double digestion respectively, reclaim big fragment, handle with the Semen Phaseoli radiati Germinatus nuclease, the excision protruding terminus, T4DNA ligase flush end connects, transformed into escherichia coli JM109 bacterium, select positive colony, enzyme is cut evaluation, obtains to have in HSAcDNA terminal deletion CTT (leucine) back the plasmid pD3HSAIFN α 1b0 with the direct fusion gene of each IFN cDNA, pD3HSAIFN α 2b0, pD3HSAIFN α 2a0, pD3HSAIFN β 0 etc.
Equally, can add multiple flexible joint, as [Gly Gly Gly Gly Ser] at HSA and IFN
n, n can be 1-10.
As using [Gly Gly Gly Gly Ser]
2During as joint, available synthetic oligonucleotide:
L2a:5’CCTTGGTGGTGGCGGTTCCGGCGGTGGTG3’
L2b:5’GATCCACCACCGCCGGAACCGCCACCACCAAGG3’
As using [Gly Gly Gly Gly Ser]
3During as joint, available synthetic oligonucleotide:
L3a:5’CCTTGGTGGTGGCGGTTCCGGCGGTGGTGGCTCCGGTGGCGGCG3’
L3b:5’GATCCGCCGCCACCGGAGCCACCACCGCCGGAACCGCCACCACCAAGG3’
With plasmid pD3HSAIFN α 1b, pD3HSAIFN α 2b, the pD3HSAIFN β that makes up among the embodiment 4, use BamH I/Stu I double digestion respectively, reclaim big fragment, oligonucleotide L2a is connected with the big fragment that reclaims with L2b annealing back, transformed into escherichia coli JM109, the picking positive colony, enzyme is cut evaluation, obtains to have between HSAcDNA end and each IFN cDNA to insert coding [Gly Gly Gly Gly Ser]
2Plasmid pD3HSAIFN α 1b2, the pD3HSAIFN α 2b2 of the oligonucleotide of flexible joint, (its step as shown in Figure 2) such as pD3HSAIFN α 2a2, pD3HSAIFN β 2.
Equally with plasmid pD3HSAIFN α 1b, the pD3HSAIFN α 2b, pD3HSAIFN α 2a, the pD3HSAIFN β that make up among the embodiment 4, use BamH I/Stu I double digestion respectively, reclaim big fragment, oligonucleotide L3a is connected with the big fragment that reclaims with L3b annealing back, the transformed into escherichia coli e. coli jm109, the picking positive colony, enzyme is cut evaluation, obtains to have between HSAcDNA end and IFN cDNA to insert coding [Gly Gly Gly Gly Ser]
3Plasmid pD3HSAIFN α 1b3, the pD3HSAIFN α 2b3 of the oligonucleotide of flexible joint, pD3HSAIFN α 2a3, pD3HSAIFN β 3 etc.Available similar methods is transformed pPIC9 IFN α 1b HSA, the plasmids such as pPIC9 IFN α 2b HSA, pPIC9 IFN β HSA that embodiment 5 makes up, to insert different joints between IFN and HSA.
Plasmid with above structure, as pD3HSAIFN α 1b0, pD3HSAIFN α 2b0, pD3HSAIFN α 2a0, pD3HSAIFN β 0 pD3HSAIFN α 1b2, pD3HSAIFN α 2b2, pD3HSAIFN α 2a2, pD3HSAIFN β 2, pD3HSAIFN α 1b3, pD3HSAIFN α 2b3, pD3HSAIFN α 2a3, pD3HSAIFN β 3 grades use the method identical with embodiment 4 to make up corresponding plasmid pHIL-D2HSAIFN α 1b0 respectively, pHIL-D2HSAIFN α 2b0, pHIL-D2HSAIFN α 2a0, pHIL-D2HSAIFN β 0, pHIL-D2HSAIFN α 1b2, pHIL-D2HSAIFN α 2b2, pHIL-D2HSAIFN α 2a2, pHIL-D2HSAIFN β 2, pHIL-D2HSAIFN α 1b3, pHIL-D2HSAIFN α 2a3, pHIL-D2HSAIFN α 2b3, pHIL-D2HSAIFN β 3, transform pichia spp GS115, obtain to express respectively the HSAIFN α 1b0 that directly merges with each IFN behind the HSA terminal deletion leucine, HSAIFN α 2b0, HSAIFN α 2a0, HSAIFN β 0 fusion rotein such as grade, insert the HSAIFN α 1b2 of [Gly Gly Gly Gly Ser Gly Gly GlyGly Ser] flexible joint between HSA and IFN, HSAIFN α 2b2, HSAIFN α 2a2, HSAIFN β 2 fusion roteins such as grade and HSA and IFN insert the fusion rotein HSAIFN α 1b3 of [Gly Gly Gly Gly Ser GIy Gly Gly Gly Ser Gly Gly Gly GlySer] flexible joint, HSAIFN α 2b3, HSAIFN α 2a3, HSAIFN β 3 etc.
Embodiment 7: the purifying of fermentation of engineering zymic and fusion rotein
The engineering yeast fermentation of the expression HSA/IFN α 2b fusion rotein that makes up with embodiment 5 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
2O0.15g/L, K
2SO
42.4g/L, MgSO
4.7H
2O1.95g/L, KOH0.65g/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
2O6.0g/L, CoCl
2.6H
2O, MnSO
4.H
2O3.0g/L, H
3BO
30.02g/L, FeSO
4.7H
2O65.0g/LNaMoO
4.2H
2O0.2g/L, ZnSO
4.7H
2O20.0g/L, KI0.1g/L, dense H
2SO
45ml/L, 121 ℃ of autoclavings 30 minutes), 0.5ml/L0.02% 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, the 2ml/L0.02% vitamin H) and induces.Inducing culture 60 hours.Get the 50ml medium centrifugal and get supernatant, add ammonium sulfate to 20% saturation ratio, 4 ℃ of placements are spent the night behind the mixing, the centrifuging and taking precipitation, dissolve with 3ml25mmol/LTris-HCl (pH7.0), with Supdex 200 (Amersham Pharmacia Biotech UK) Φ 1.6 * 100cm column chromatography for separation, moving phase is 25mmol/LTris-HCl (pH7.0), 150mmol/LNaCl, flow velocity is 1ml/L, ultraviolet detection is collected first peak (retention time is 70-85 minute), is the fusion rotein of purifying, molecular weight be 85KD (as shown in Figure 5, as shown in Figure 5, wherein, the 1st, molecular weight standard, be respectively 94 from top to bottom, 67,43,30KD; The 2nd, the HSAIFN α 2b of purifying).Purification of samples with cytopathic-effect inhibition assay measure the IFN activity (method is seen " Chinese biological goods rules " 2000 editions, and the Chinese biological standard of articles council compiles, Chemical Industry Press 2000.6, p373-375).Wish cell (human amniotic cell) is incubated in the MEM substratum that contains 10% calf serum, go down to posterity weekly and (use trysinization with preceding, be made into the MEM substratum that contains 10% calf serum for 2 times.2.5-3.5 * 10
5Individual cell/ml).Be inoculated in the 96 porocyte culture plates every hole 100 μ l, 37 ℃, 5%CO
2Cultivated 4-6 hour.In another 96 porocyte culture plate, every hole adds 150 μ l and measures nutrient solution (the MEM substratum that contains 7% calf serum), and sample is made 2 times of gradient dilutions, and every hole 100 μ l sample diluting liquids are transferred in the culture plate that is added with cell, and 37 ℃, 5%CO
2Cultivated 18-24 hour, and abandoned supernatant, add and use the MEM substratum that contains 3% calf serum to be diluted to 100TCID
50Vesicular stomatitis virus (VSV virus), every hole 100 μ l, 37 ℃, 5%CO
2Cultivated 24 hours, and abandoned supernatant, every hole adds 50 μ l staining fluids (50mg Viola crystallina is dissolved in 20% ethanol), room temperature was placed 30 minutes, flowing water carefully washes away staining fluid, blots residual moisture, and every hole adds 100 μ l destainers (0.1% acetate, 50% ethanol), room temperature was placed 5 minutes, measure its absorbance at 570nm, as shown in Figure 6, transverse axis is the hole count of 2 times of gradient dilutions, the longitudinal axis is an absorbance, and show sample obviously has interferon activity as a result.
With the sample bag by elisa plate, anti-with rabbit AHS albumin antibody respectively as one, the goat anti-rabbit igg of horseradish peroxidase-labeled is anti-as two, test sample, as shown in Figure 7, transverse axis is the hole count of 2 times of gradient dilutions, and the longitudinal axis is an absorbance, as can be seen from the figure, sample has the antigenicity of HSA.
Get HSA/IFN α 2b, HSA/IFN beta fusion proteins and rhIFN α 2b, the rhIFN β of identical active dose, the mouse tail vein administration, different time is got determination of serum IFN activity wherein.As shown in Figure 8, the accretion rate of fusion rotein in mice plasma is obviously slow than rhIFN.Injected back 10 minutes, both are more or less the same at the IFN activity in blood plasma, and after 8 hours, the activity of rhIFN α 2b and rhIFN β has descended nearly ten times, and the activity of fusion rotein has only reduced slightly; After 48 hours, the mouse of injection fusion rotein still can measure the activity of IFN.