CN101942026A - Long-acting interferon fusion protein and application thereof - Google Patents
Long-acting interferon fusion protein and application thereof Download PDFInfo
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Abstract
The invention relates to the field of biological pharmacy, in particular to a recombinant long-acting interferon fusion protein and application thereof. The technical problem to be solved is to improve internal action time of the interferon and provide a fusion protein. The fusion protein consists of an N-end interferon, C-terminal human albumin binding peptide and glycin linking peptide which links between the N-end interferon and the C-terminal human albumin binding peptide. The protein can be expressed efficiently in colon bacillus, is simple in purification process and can be used for large-scale industrial production and preparation of a pharmaceutical grade rcIFN-ABP fusion protein pure product. Compared with the recombinant cognate interferon (rcIFN) per se, the rcIFN-ABP fusion protein obtained by using the invention has the characteristic of longer blood serum half-life period. Meanwhile, the specificity antivirus ratio activity is equivalent to cIFN. The rcIFN-ABP fusion protein can be used for treating virus infectious diseases, such as AIDS, hepatitis, SARS, bird flu and the like, and also can be used for treating tumors and other cell proliferative diseases.
Description
Technical field:
The present invention relates to field of biological pharmacy.Be specifically related to a kind of recombinant long-acting homology Interferon, rabbit (cIFN)-people's protein binding peptide (ABP) fusion rotein that is used for the treatment of viral infection disease and tumour and cell proliferation disorders, and uses thereof.
Background technology:
People have found that a kind of albumen as antiviral substance is referred to as Interferon, rabbit (interferon) in early days when the interference phenomenon (virus interference) of research virus.The chicken cell of nineteen fifty-seven Isaacs and Lindenmann proof influenza infection can be secreted a kind of soluble factor mediated cell homology and allos virus are all produced resistant function.Nagano also observed and had obtained similar result with Kojima in 1958.These all are to illustrate the living organism interferon system afterwards in more detail to have laid early stage basis.In the 1970's, from white corpuscle, be purified into interferon protein and just make Interferon, rabbit really can be used for studying purposes.1980, owing to successfully can obtain the large-scale purification interferon formulation with gene engineering method, people can carry out clinical experiment and treatment to virus infection and tumour patient by enough Interferon, rabbit like this.
The multigene family albumen that Interferon, rabbit is made up of one group of cytokine of inducing generation.Interferon, rabbit has extensive effect, and it is adjustable to comprise a series of biological functions such as virus multiplication.Interferon, rabbit is divided into two kinds usually: I type Interferon, rabbit, the malicious Interferon, rabbit of also pretending illness (Viral IFNs) comprises alpha-interferon (LeIF) and beta-interferon (fiblaferon), and ω-Interferon, rabbit.II type Interferon, rabbit also claims type II interferon (gamma-interferon).Viral interferon is induced generation by virus infection.II type Interferon, rabbit is then induced generation by mitogenesis or antigenic stimulation.The cell of most of virus infection can synthesize interferon-alpha/beta in cultivation.Yet gamma-interferon can only synthesize it by immune active cells, comprises natural killer cell (NK), CD4
+Th
1Cell and CD8
+Born of the same parents' poison/suppressor T cell.
Human body has many viral interferon encoding genes, comprises 14 kinds of alpha-interferon genes, a kind of beta-interferon and a kind of ω-interferon gene.They do not contain intron (intron), and they are arranged in together at people's No. 9 the short arm of a chromosome.Mouse is then at its No. 4 karyomit(e) respective regions.Gamma-interferon has only a kind, and it has 3 introns, and it is long-armed that it is positioned at people's No. 12 karyomit(e); Mouse then is No. 10 karyomit(e).Although some Interferon, rabbit can be glycosylation modified through modification such as N-and O-after some translations in body, main human is not by glycosylation.Alpha-interferon is mainly brought into play its biological effect with monomer, and beta-interferon and gamma-interferon then work with homologous dimerization source bodily form formula.Do not know also now why human body has so many alpha-interferon genes.When removing the single beta-interferon gene of mouse with gene knockout (gene knock-out) method, the mouse that produces is then very responsive to virus infection, the numerous alpha-interferon genes of this explanation can not compensate the effect of beta-interferon gene, and beta-interferon has the effect of its uniqueness in process of resisting virus infection.Owing in the laboratory Interferon, rabbit has been carried out a large amount of successful fundamental researchs, this has just had the application of Interferon, rabbit in clinical treatment afterwards.Three kinds of Interferon, rabbit: α of U.S. FDA approved-, β-and gamma-interferon as the clinical treatment medicine.Being used for the most widely for alpha-interferon, disease of viral infection comprises B-mode and hepatitis C.In addition, alpha-interferon also is approved for the treatment of Kaposi sarcoma, venereal disease bleb, hairy cell, chronic myelogenous leukemia and pernicious myelomatosis.Beta-interferon is approved for the treatment after the multiple sclerosis recurrence.Gamma-interferon then is approved for the treatment of chronic heredity immunologic derangement (chronic glomerulus disease).The interferon medicine that has the man biopharmaceutical company of number production for treating to use.The interferon-' alpha ' 2a of the Hoffman-La Roche manufacturer of company product RoferonA by name; The interferon-' alpha ' 2b of the manufacturer of Schering company product Intron A by name; The homology Interferon, rabbit of the manufacturer of Amgen company name of an article Infergen; Interferon Sciences company has produced the interferon-' alpha ' N3 that is prepared from by human leukocyte of commodity Alferon N by name; Glaxo Wellcome company has produced the beta-interferon of commodity Avonex by name, Chiron company commodity Betaseron by name.Genentech company has produced the gamma-interferon of trade(brand)name Actimmune.January calendar year 2001, drugs approved by FDA Peg-Intron-α 2b of the trade(brand)name PEG-Intron that produces of Schering company be used for the treatment of the chronic hepatitis C patient who did not accept interferon therapy before this.Roche also produces Pegylation and gets interferon-' alpha ' 2a (commodity are called Pegasys) and be used for the treatment of the chronic hepatitis C patient.
(Consensus Interferen cIFN) is a complete synthesis I type Interferon, rabbit to the homology Interferon, rabbit.By analyzing relatively 14 kinds of natural alpha-interferon subtype protein matter sequential structures, the amino acid of choosing on corresponding each position of different subtype the most normal appearance is formed a new protein sequence (being homologous sequence) thereby is obtained a brand-new homology Interferon, rabbit.Compared the effect aspect antiviral, inhibition of cell proliferation, activation NK cytoactive, cytokine induction and interferon-stimulated gene expression activity of homology Interferon, rabbit and natural interferon.Aspect all these effects, with interferon-' alpha ' 2a and-α 2b compares, the effect of homology Interferon, rabbit is all strong than the two.This may be owing to cell surface I type Interferon Receptors is formed the reason higher than alpha-interferon avidity to the homology Interferon, rabbit.On the contrary, a little less than inducing aspect the il-1 β generation homology Interferon, rabbit than alpha-interferon.These results also may reflect the result with the different keying actions of the interactional multiple collaborative albumen of I type Interferon Receptors (accessery proteins).These unique biological characteristics make the homology Interferon, rabbit have a clear superiority on clinical treatment than natural generation and recombinant natural Interferon, rabbit.
Clinical trial proof homology Interferon, rabbit can be used for treating a series of diseases and comprises chronic viral infection and some malignant tumour.In various safety of carrying out at a plurality of clinical experiments center and the validity experiment the chronic hepatitis of high titre hepatitis C virus having been infected in homology Interferon, rabbit and lymphocyte source property alpha-interferon [IFN-α (NAMALWA)] treatment compares.The validity of homology Interferon, rabbit (18MIU) obviously is better than IFN-α (NAMALWA) (9MIU) under the situation that does not increase the additional toxicity effect; Particularly when the patient when to have disturbed genotype be 1b viral effect more remarkable.The homology Interferon, rabbit can act on the failure of treatment chronic hepatitis C patient and conventional interferon therapy safely, effectively and treat the patient that recur the back.Recently the homology Interferon, rabbit also share in treatment chronic hepatitis C patient with other anti-liver class chemicals such as ribavirin.The homology Interferon, rabbit also has been successfully used to hepatitis B patient's treatment.
Yet, interferon therapy is the same with many protein drugs have obvious weak point be exactly transformation period in the blood short, alpha-interferon is eliminated it rapidly after being injected into body.Under this short circulating half-life condition,, often adopt multiple-effect amount (every day or inferior on every Wendesdays) long-time medication (6-12 month or longer) in order to reach result of treatment effectively.In order to improve the pharmacokinetics of alpha-interferon, reduce medicine frequency.The interferon medicine (Pegglated IFN) that some drugmaker combines the peg molecule of alpha-interferon and-12-KDa or 30-KDa size to have prepared a kind of moulding that narrows.This Peg-Intron molecule can make alpha-interferon prolong action time in vivo, and its front clinical therapeutic efficacy is arranged.It is obviously relevant with the composition and the binding site of bonded peg molecule that the physical chemistry of Peg-Intron and biological characteristics research have been disclosed the special antiviral activity of this semi-synthetic interferon molecule.This Pegylation effect can reduce the specific biological of alpha-interferon than living.
Albumin (molecular weight is about 67KDa) is rich in protein in the blood, and concentration is 50mg/ml (600um).In the human transformation period is 19 days.Albuminous function comprises keeps the blood plasma pH value, and oncotic pressure makes and send meta-bolites, lipid acid etc.And it can act on the main pharmaceutical carrier albumen in the blood plasma.Human albumin (HSA) the long half-lift and stability make it provide the fugitive treatment albumen of a kind of ideal carrier and some to form fusion molecule.Can make latter's transformation period increase in the rat body reach 5 hours for 3 times the albumin bound functional zone of staphylococcal protein G and people's complement receptor-1 composition fusion rotein.Alpha-interferon and albumin are merged a kind of brand-new fusion protein molecule of generation.Thereby human genome company is cascaded beta-interferon and HSA encoding gene and gives expression to a kind of the two fusion rotein (Albuferon-β). this recombination fusion protein has antiviral and the inhibition of cell proliferation activity, can start the signal conduction of IFN-stimulated response element (ISRE) mediation.Yet the specific biological specific activity of this fusion rotein is lived still low than alpha-interferon.This may be because HSA and interferon molecule covalency can influence the latter's space conformation when merging.
Prove and also can prolong some fugitive proteic transformation period with non-covalent combination of albumin.But at present not about the correlative study of Interferon, rabbit, and how carrying out real Interferon, rabbit, to carry out effective non-covalent the combination with albumin also be unknown at present.
Summary of the invention
The technical problem to be solved in the present invention is can also keep its activity as much as possible in the transformation period in the body that prolongs Interferon, rabbit.
The technical scheme that the present invention solves the problems of the technologies described above provides a kind of long-acting interferon fusion rotein.This long-acting interferon fusion rotein is to connect the affine small peptide of human albumin specificity (HSA-BP) by the C-terminal of Interferon, rabbit by one section connection peptides to form.
Preferably, the Interferon, rabbit in the above-mentioned long-acting interferon fusion rotein is the homology Interferon, rabbit.
Wherein, the aminoacid sequence of above-mentioned homology Interferon, rabbit is shown in the SEQ ID NO.1.
H2N-MCDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWDESLLEKFYTELYQQLNDLEACVIQEVGVEETPLMNVDSIIAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSTNLQERLRRKE-COOH(SEQ?ID?NO.1)。
Wherein, the aminoacid sequence of the affine small peptide of human albumin specificity (HSA-BP) in the above-mentioned long-acting interferon fusion rotein is shown in the SEQ ID NO.2.
H2N-SQRLMEDICLPRWGCLWEDDF-COOH(SEQ?ID?NO.2)。
Wherein, its aminoacid sequence of above-mentioned long-acting interferon fusion rotein is shown in the SEQ ID NO.3.
H2N-MCDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWDESLLEKFYTELYQQLNDLEACVIQEVGVEETPLMNVDSIIAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSTNLQERLRRKEGGGSQRLMEDICLPRWGCLWEDDF-COOH(SEQ?ID?NO.3)。
The aminoacid sequence of the connection peptides between the affine small peptide HSA-BP of the C-terminal of Interferon, rabbit wherein and human albumin specificity is GGG.
The present invention also provides the nucleotide sequence of the long-acting interferon fusion rotein of encoding above-mentioned when above-mentioned long-acting interferon fusion rotein is provided.
Further, the nucleotides sequence of code book invention long-acting interferon fusion rotein is classified as shown in the SEQ ID NO.4.
ATGTGCGACCTGCCGCAGACCCACTCCCTGGGTAACCGTCGTGCTCTGATCCTGCTGGCTCAGATGCGTCGTATCTCCCCGTTCTCCTGCCTGAAAGACCGTCACGACTTCGGTTTCCCGCAGGAAGAATTCGACGGTAACCAGTTCCAGAAAGCTCAGGCTATCTCCGTTCTGCACGAAATGATCCAGCAGACCTTCAACCTGTTCTCCACCAAAGACTCCTCCGCTGCTTGGGACGAATCCCTGCTGGAAAAATTCTACACCGAACTGTACCAGCAGCTGAACGACCTGGAAGCTTGCGTTATCCAGGAAGTTGGTGTTGAAGAAACCCCGCTGATGAACGTTGACTCCATCCTGGCTGTTAAAAAATACTTCCAGCGTATCACCCTGTACCTGACCGAAAAAAAATACTCCCCGTGCGCTTGGGAAGTTGTTCGTGCTGAAATCATGCGTTCCTTCTCCCTGTCCACCAACCTGCAGGAACGTCTGCGTCGTAAAGAAGGCGGTGGCTCTCAGCGTCTGATGGAAGATATCTGCCTGCCGCGTTGGGGCTGCCTGTGGGAAGATGATTTCTAA(SEQ?ID?NO.4)。
The present invention also provides the carrier that contains claim 6 or 7 described nucleotide sequences.
The present invention also provides the host cell that contains claim 6 or 7 described nucleotide sequences.
Simultaneously, the present invention also provides the purposes of above-mentioned long-acting interferon fusion rotein in preparation treatment antiviral or antitumor drug.
The present invention also provides a kind of treatment antiviral or antitumor drug, and this antiviral or antitumor drug are that main active ingredient is added pharmaceutically acceptable complementary composition and is prepared from above-mentioned long-acting interferon fusion rotein.
Adopt high-throughput screening method (HTS), found that a small peptide (HSA-BP) can optionally carry out high-affinity with human albumin and combine.The present invention design and the dna encoding sequence of having synthesized this small peptide are passed through a glycine chain encoding sequence link together with its encoding sequence with Interferon, rabbit, thus coded interference element-HSA-BP fusion rotein.HSA-BP is positioned at the C-end of Interferon, rabbit.The encoding sequence of this fusion rotein is cloned in the escherichia coli high-level expression carrier goes, can be by containing escherichia coli high-level expression Interferon, rabbit-HSA-BP (cIFN-HSA-BP) fusion rotein of expression vector.Adopt biochemical separation purification method from the intestinal bacteria lysate, to obtain highly purified cIFN-HSA-BP fusion rotein.Experiment in vitro proves that this fusion rotein disturbs the identical antiviral and inhibition of cell proliferation effect that have with homology.
It is single with 12 times of cIFN prolongations to experiment showed, that fusion rotein of the present invention is expelled to interior its transformation period ratio of mouse body.This illustrates the cIFN-ABP fusion rotein in antiviral, inhibition of cell proliferation biologic activity while with Interferon, rabbit, its transformation period significant prolongation in vivo, thus its clinical treatment effect has remarkable advantages than Interferon, rabbit itself.
Description of drawings
The segmental gel electrophoresis checking of the cIFN-GGG-ABP coding DNA of Fig. 1,0.7KB size is figure as a result.
Fig. 2, gel electrophoresis checking be figure as a result.Lane 1:1KD DNA ladder marker (1 μ g/10 μ l), Lane 2:pBAD-cIFN plasmid NheI+SphI enzyme is cut thing 20 μ l, and Lane 3:pBAD-cIFN plasmid XbaI+SacI enzyme is cut thing 20 μ l, Lane 4 and 5:PCRII reaction product 20 μ l.
Fig. 3, gel electrophoresis checking be figure as a result.1:1kb DNA ladden Marker (1 μ g/10 μ l); 2: the result of colony 1SacI+xbaI enzyme; 3: the result of colony 2SacI+xbaI enzyme; 4: the result of colony 3SacI+xbaI enzyme; 5: the result of colony 4SacI+xbaI enzyme; 6: the result of colony 5SacI+xbaI enzyme.
The ECoRV enzyme of Fig. 4, pBAD-cIFN is cut gel electrophoresis figure as a result.PBAD-cIFN plasmid after the 1:ECoRV enzyme is cut; 2: the pBAD-cIFN plasmid of cutting without enzyme; 3:1kb DNA Ladder marker (1 μ g/10 μ l).The result shows that the ECoRV enzyme is cut and can be made the pBAD-cIFN plasmid linearization.
The enzyme of Fig. 5, pBAD-cIFNm is cut Fig. 1 as a result: 1kb DNA ladder make (1 μ/10 μ l); The 2:pBAD-cIFNm# 1 plasmid is cut through the ECORV enzyme; 3:pBAD-cIFNm# 1 plasmid SacI+Xba double digestion; 4:PCR-Blunt II Topo-cIFN plasmid is through the SacI+Xbal double digestion.
The immunoblotting of Fig. 6, production purge process (Western Blot) is identified Fig. 1, is not used L-arabinose inductive bacterial lysate; 2, the bacterial lysate after L-arabinose is induced; 3, cross the sample of Q-Sepharose post; The albumen that 4 final purifications are finished.
Fig. 7, Western blot immunoblotting be figure as a result.The 1st road can be got off with the human serum albumin co-immunoprecipitation by human albumin antibody for reorganization cIFN-ABP, and the 2nd road for reorganization cIFN co-immunoprecipitation does not take place.
Fig. 8, reorganization cIFN-ABP fusion rotein combine test-results with other animal serum albumin.1 human albumin is cooked contrast; 2: the dog albumin; 3: rat albumin; 4: the monkey albumin; 5: the rabbit albumin; 6:: mouse albumin.
Embodiment
Further specify by concrete enforcement formula below in conjunction with accompanying drawing but do not limit the present invention.
The acquisition of embodiment one reorganization cIFN-ABP protein coding gene and the structure of efficient expression engineering thereof
Artificial chemosynthesis cIFN coding gene sequence, and be cloned in the pBAD18 plasmid vector (available from the handsome Invitrogen of company, Carlsbad, USA, its sequence is referring to J.Bacteriol.177,4121-4130 (1995)), pBAD-cIFN obtained.
Be template at first, add the affine small peptide of human albumin specificity (HSA-BP is called for short ABP) encoding sequence and catenation sequence at its C-end with PCR method with the cIFN encoding sequence in this plasmid.The pBAD18 plasmid is the escherichia coli high-level expression plasmid.The cIFN encoding sequence is the XbaI and the SacI restriction enzyme site place of being cloned into this plasmid.Therefore, the encoding sequence that divided for 2 steps added ABP, connection peptides and restriction enzyme site with PCR method so that its cIFN-ABP fusion rotein encoding sequence can be cloned in this pBAD18 expression vector again goes to efficiently express.
PCR reacts I: introduce the encoding sequence of connection peptides (GGG) and ABP (SQRLMEDICLPRW GCLWEDDF) at cIFN encoding sequence C-terminal, in the 0.2mlPCR pipe, add the following ingredients mixing it:
PCR is reaction time:
I:98 ℃ 2 minutes
II:25 cycle
III:72 ℃ 5 minutes
IV:4 ℃ standby
With reactant electrophoresis in 1% agarose gel.The result is as shown below, as the unanimity that predicts the outcome, and has obtained the cIFN-GGG-ABP coding DNA fragment (the gel electrophoresis checking the results are shown in Figure 1) of a 0.7KB size.
This 0.7kb size reclaimed to be purified into from agarose gel be used as the template of PCR next time, be used for follow-up clone to introduce the XbaI enzyme cutting site in 3 ' end downstream.
PCR reacts II: add following composition and mixing in the PCR of 0.2ml pipe:
PCR reaction time is with aforementioned PCR reaction time.
Then the pBAD-cIFN plasmid is carried out the XbaI+SacI double digestion:
In 37 ℃ of incubations 2 hours.
Simultaneously this plasmid is used NheI and SphI double digestion in contrast:
37 ℃ of incubations 2 hours.
The electrophoresis (the gel electrophoresis checking the results are shown in Figure 2) in 1% agarose gel with plasmid enzyme restriction reactant and PCR product.PBAD plasmid vector dna fragmentation after the PCRII reaction product dna fragmentation of Lane4 and Lane 3XbaI-SacI enzyme cut reclaims from agarose gel, purifying.
The PCRII reactant is cloned into pCR-BLUNTII ToPo carrier, and (lnvitrogen goes in USA).
React as follows:
The reactant mixing is put 20 ℃ of reactions 10 minutes.
Then reactant is transformed Mach1 (Invitrogen) competence bacterial cell.Transform bacteria is applied in the agarose plate of LB+Amp (100 μ g/ml), puts 37 ℃ of overnight incubation.Select single bacterium colony next day, the bacterium of in the LB substratum, increasing, preparation plasmid DNA (Qiagen Miniprep Kit).Plasmid DNA is carried out the double digestion Screening and Identification with XbaI and SacI.Endonuclease reaction is:
37 ℃ of incubations 2 hours.
The endonuclease reaction thing is joined electrophoresis in 1% agarose gel, the result as shown in Figure 3:
Fragment Separation and Recovery purifying from agarose with the SacI-XbaI enzyme of No. 5 colony 0.7kb sizes among Fig. 3.This fragment contains the cIFN-GGG-ABP encoding sequence.
At last, the SacI-XbaI dna fragmentation that this is contained the cIFN-GGG-ABP encoding sequence is cloned in the pBAD18 SacI-XbaI restriction enzyme site and is gone.The DNA ligation is as follows:
20 ℃ of ligations 4 hours.
As previously mentioned, (Invitiogen USA), is applied to transform bacteria on LB+Amp (100 μ g/ μ l) the agarose culture plate, measures 37 ℃ and cultivates liquid this DNA ligation thing to be transformed MachI competence bacterial cell.Arrange the menu colony inoculation bacterium of in 2ml LB+Amp (100 μ g/ml) substratum, increasing next day.Use Qiagen Mini-Prep kit extracting and purifying bacteria plasmid DNA then, plasmid DNA is carried out enzyme cut evaluation.Earlier use SacI, the XbaI double digestion filters out the positive colony that contains PCRII SacI-XbaI insertion sequence.
With the pBAD plasmid called after pBAD-cIFNm that contains the cIFN-ABP insertion sequence that filters out, further pBAD-cIFN and pBAD-cIFNm plasmid are carried out the comparison of restriction enzyme mapping, in the pBAD-cIFN plasmid, only contain an ECoRV restriction enzyme site, therefore, cut with the ECoRV enzyme and can make this plasmid linearization, the ECoRV enzyme is cut the result as shown in Figure 4.The result shows that the ECoRV enzyme is cut and can be made the pBAD-cIFN plasmid linearization.
Then, choose 1 pBAD-cIFNm plasmid clone and carry out SacI+XbaI double digestion and ECoRV single endonuclease digestion respectively.Method is the same.Owing to itself contain an ECoRV restriction enzyme site except the pBAD18 plasmid, so pBAD-cIFNm cuts through the ECoRV enzyme and can produce 2 dna fragmentations, the result as shown in Figure 5, fully with predict the outcome consistent.
All results show that pBAD-cIFNm contains correct restriction enzyme site and cIFN-ABP insertion sequence.
At last, pBAD-cIFNm is carried out the dna sequencing analysis.The result shows that all cIFN, connection peptides and ABP encoding sequence and implementation sequence meet fully, have reached purpose of design.
Embodiment two, rcIFN-ABP are proteic to be efficiently expressed and purifying
TOP10 competence bacterium is available from American I nvitrogen company.The pBAD-IFNm plasmid is changed in the TOP10 bacterial cell according to a conventional method, transform bacteria is inoculated on the LB agarose culture plate (containing 100 μ g/ml penbritins), put 37 ℃ of overnight incubation.Get single colony inoculation in 5ml LB liquid nutrient medium (containing 100 μ gml penbritins), put in 37 ℃ of shaking tables and to cultivate (225-250RPM) and spend the night.Get the 1ml bacterial cultures that spends the night and join in the 100ml LB liquid nutrient medium (containing 100 μ g/ml penbritins), put 37 degree shaking tables and cultivate, when OD600=～0.5, add the L-arabinose of 1ml 20%, continue to cultivate 4-8 hour.The bacterial cell of results after inducing, with bacterial cultures in Sorvall whizzer (RC-5C, Dupont, USA, rotary head model SS-34) in 4 ℃, centrifugal 15 minutes of 6000RPM.(10mM Tris-HCl, pH7.2,10mM EDTA, 100mM NaCl) washes the bacterial precipitation piece once with damping fluid.Then the bacterial precipitation thing is suspended in 100ml Tris damping fluid (50mM Tri-HCl, pH8.5,5mM EDTA, 1mM PMSF).Place ultrasonication bacterium (30 seconds fragmentation/30 second cooling/200-300Wx30 cycles).Place centrifugal 30 minutes of 4 ℃ of 12000RPM to obtain inclusion body precipitation piece bacterial lysate.Inclusion body is precipitated piece be suspended in the aforementioned Tris damping fluid (adding 1%deoxycholic acid Septochol), wash 2 times to remove impurity.Then the inclusion body throw out is washed once with the Tris damping fluid separately.At last more once with Milli Q washing.
Inclusion body is dissolved in the Tris/ urea buffer solution of high pH (50mM Tris-HCl, pH12.5,5mM EDTA, 1Mm PMSF, 2M urea).Constantly concussion is so that its dissolving.General 0.5g inclusion body is dissolved in the 20ml damping fluid.Solution is shaken up 5 minutes in room temperature, then, put centrifugal 15 minutes of 12000RPM to remove insoluble cell impurity.Get the supernatant that contains inclusion body and dropwise add (50mMTris-HCl, pH8.0,1mM EDTA, 2M urea, 10% sucrose, 1mM PMSF, 0.1mM oxidized form and 1mM reduced glutathion (glutamthiones), stirring in the renaturation buffer while dripping.The renaturation buffer volume is 10 times of adding inclusion body solution.Place 4 ℃ to stir 4 hours this solution to allow its albumen begin the part renaturation.Measure the pH of solution and be transferred to pH8.4.With this solution at damping fluid (50mMTri s-HCl, pH8.4,1mM EDTA, 2mM urea, 10% sucrose, 1mMPMSF) dialysis.Every dialysis is after 3 hours, again at following damping fluid dialysis (0.5mM EDTA, 2.5% sucrose, 1mM PMSF gradually reduces wherein urea content 2M, 1.5M, 1M, 0.5M, 0 for 20mMTris-HCl, pH8.4), totally 5 dialysis, each 3 hours.
Recombinant protein solution was put 4 ℃ of centrifugal 12000RPM 30 minutes, get supernatant through 0.45 μ m membrane filtration.Protein solution after filtering is splined on Q-Sepharose post (50ml) carries out purifying.Q-Sepharose before used damping fluid (50mMTris-HCl, pH8.4,1mM EDTA, 5% sucrose, 1mM PMSF) balance it.Sample speed is 0.5ml/ minute on the albumen.Then post is washed (20 times of column volumes, 1ml/ minute speed) with level pad.The albumen that is combined on the post carries out wash-out with 0～1M NaCl salt gradient, and speed is 1ml/ minute.Every pipe 2ml collects, with the protein peak of UV detector monitoring collection.With protein example in the SDS-PAGE electrophoretic analysis.And carry out immunoblotting (Western Blot) with anti-cIFN interferon antibody and identify it (the results are shown in Figure 6).The collection tube that will contain the cIFN-ABP fusion rotein merges together, and carries out PBS dialysis (4 ℃, 4 hours * 3) then. the protein sample after the dialysis put-70 ℃ frozen.
The detection of test example one, reorganization cIFN-ABP antiviral activity and with the comparison of cIFN
With the A549 cell with 1 * 10
4Cells/well joins in 96 well culture plates, and substratum is DMEM+10% foetal calf serum (Sigma).After 24 hours, add Interferon, rabbit, continue to cultivate 24 hours.Remove Interferon, rabbit, add EMCV virus (1pfu/ cell) solution (DMEM+2% foetal calf serum) cells infected, cultivated 1 hour.Remove virus, add fresh culture (DMEM+10% foetal calf serum) and continue to cultivate 24 hours, use Cell Biolabs then, the Cytoselect that Inc produces
TM(San Diego, CA USA) measure the viable cell survival rate to cell Viabilityand cytotoxicity Assay test kit.Mark the EC50 value according to the plain dosage one response curve meter of interference.
Table 1, antiviral activity and the comparison thereof of reorganization cIFN-ABP in the A549 cell
| The interference element | EC50(pg/ml) |
| rc?IFN-ABP | 4.8 |
| rcIFN | 4.4 |
| rIFN-α2b | 5.8 |
This shows that reorganization cIFN-ABP is suitable with reorganization cIFN antiviral activity, the two all is higher than the antiviral activity of reorganization IFN-α 2b.
The test that combines of test example two, reorganization cIFN-ABP and human serum albumin
With 0.5ml human serum albumin (10 μ g/ml) solution (preparing among the PBS) and 0.5ml reorganization cIFN-ABP (10 μ g/ml) or reorganization cIFN (10 μ g/ml) (all being formulated among the PBS) mixing, put 37 ℃ of incubations 30 minutes, and added anti-human albumin antibody (100 μ g/200 μ l) 20 μ l and continued incubation 1 hour.After the taking-up, add G albumen-agarose reagent, put room temperature 2 hours (maintenance whipped state).Centrifugal then removal supernatant is given a baby a bath on the third day after its birth the agarose throw out time with the PBS+0.05%Tween20 damping fluid, will add 100 μ l, 1 * laemmli damping fluid in the throw out at last, puts in the boiling water 5 minutes.After the taking-up supernatant is joined 15%SDS-PAGE and go up the sample electrophoresis, carry out Western blot western blot test then.Remove to survey nitrocellulose membrane (it is 1: 1000 that antibody is used concentration) after the transfer with anti-cIFN monoclonal antibody, the result as shown in Figure 7.
Test-results shows: reorganization cIFN-ABP can be got off with the human serum albumin co-immunoprecipitation by anti-human albumin antibody, and the two combines to prove them.Reorganization cIFN albumen then can not be with the human serum albumin co-precipitation, and proving does not have combination between them.
Test example three, reorganization cIFN-ABP fusion rotein combine test with other animal serum albumin
Mouse, rabbit, monkey, rat, dog serum albumin and agarose is crosslinked.Then agarose-animal albumin is formulated in that protein content is 10 μ g/ml among the PBS.Get albuminous solution of 0.5ml agarose-animal and 0.5ml reorganization cIFN-ABP (10 μ g/ml) mixing, put 37 ℃ and stirred incubation 30 minutes.Taking out the back washs 3 times with the PBS+0.05%Tween20 damping fluid.Adding 100 μ l 1x Laemmli damping fluids in throw out put in the boiling water 5 minutes.After the taking-up supernatant joined 15%SDS-PAGE and go up the sample electrophoresis, carry out immunoblotting (Westerm blot) test then, remove to survey nitrocellulose membrane (antibody application concentration is 1: 1000) after the transfer with anti-cIFN monoclonal antibody.The result as shown in Figure 8, the reorganization cIFN-ABP can get off with all animal albumin bound and by the agarose solids precipitation.
Clearance test in the mouse body of test example four, reorganization cIFN-ABP
Respectively through the rcIFN-ABP100 μ g/kg of tail vein injection 100 μ l volumes, rcIFN protein 10 0 μ g/kg and PBS contrast damping fluid with three groups of female Balb/c mouse (7-8 age in week).Injection back different times is from tail vein blood sampling.The antiviral activity of its blood sample adopts aforementioned identical method to measure it.The antiviral activity that the antiviral activity of its experimental group blood sample deducts corresponding PBS control group blood sample equals external source and is injected in the antiviral activity of interference element in the circulation blood sample and the results are shown in Table 2.The result shows that its transformation period is far longer than the common interference element of not transforming.
Clearance test result in the table 2 mouse body
Above-mentioned test-results proves that fusion rotein of the present invention disturbs the identical antiviral and inhibition of cell proliferation effect that have with homology.And fusion rotein of the present invention is expelled to interior its transformation period ratio list of mouse body and prolongs greatly with cIFN.This illustrates the cIFN-ABP fusion rotein in antiviral, inhibition of cell proliferation biologic activity while with Interferon, rabbit, its transformation period significant prolongation in vivo, thus its clinical treatment effect has remarkable advantages than Interferon, rabbit itself.
Claims (10)
1. long-acting interferon fusion rotein is characterized in that: connect the affine small peptide HSA-BP of human albumin specificity by the C-terminal of Interferon, rabbit by connection peptides and form.
2. long-acting homology interferon fusion protein according to claim 1 is characterized in that: described Interferon, rabbit is the homology Interferon, rabbit.
3. long-acting homology interferon fusion protein according to claim 2 is characterized in that: the aminoacid sequence of described homology Interferon, rabbit is shown in the SEQ ID NO.1.
4. long-acting homology interferon fusion protein according to claim 2 is characterized in that: the aminoacid sequence of the affine small peptide of described human albumin specificity is shown in the SEQ ID NO.2.
5. according to claim 2,3 or 4 each described long-acting interferon fusion roteins, it is characterized in that: its aminoacid sequence is shown in the SEQ ID NO.3, and the aminoacid sequence of the connection peptides between the affine small peptide HSA-BP of the C-terminal of Interferon, rabbit wherein and human albumin specificity is GGG.
6. the nucleotide sequence of coding claim 1～5 each described long-acting interferon fusion rotein.
7. the nucleotide sequence of coding long-acting interferon fusion rotein according to claim 6 is characterized in that: its sequence is shown in the SEQ ID NO.4.
8. the carrier that contains claim 6 or 7 described nucleotide sequences.
9. the host cell that contains claim 6 or 7 described nucleotide sequences.
10. each described long-acting interferon fusion rotein of claim 1～5 is preparing the purposes for the treatment of in antiviral or the antitumor drug.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012048653A1 (en) * | 2010-10-14 | 2012-04-19 | 成都正能生物技术有限责任公司 | Long-life interferon fusion protein and use thereof |
| CN102775502A (en) * | 2012-08-16 | 2012-11-14 | 天津禹王生物医药科技有限公司 | Alpha-interferon fusion protein |
| CN104583240A (en) * | 2012-08-13 | 2015-04-29 | Jw可瑞基因株式会社 | Interferon-[alpha] fusion protein in which cytoplasmic transduction peptide and polyethylene glycol are bonded to one another |
| CN106729635A (en) * | 2016-12-19 | 2017-05-31 | 武汉市疾病预防控制中心 | A kind of applications of IFN λ 3 in prevention or treatment AIDS-treating medicine is prepared |
| CN106967175A (en) * | 2013-05-15 | 2017-07-21 | 复旦大学 | Long-acting interferon and its production and use |
| CN116333166A (en) * | 2023-02-08 | 2023-06-27 | 珠海臻谱基因科技有限公司 | Recombinant interferon targeting HIV gp120 protein and application thereof |
| CN117430716A (en) * | 2023-10-16 | 2024-01-23 | 珠海臻谱基因科技有限公司 | Recombinant interferon drug targeting conserved HIV gp41 subunit near-membrane-end outer region and application thereof |
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| CN1740197A (en) * | 2004-08-26 | 2006-03-01 | 辉阳科技美国公司 | Recombination interferon with new space conformation and enhanced effect, its preparing method and application |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012048653A1 (en) * | 2010-10-14 | 2012-04-19 | 成都正能生物技术有限责任公司 | Long-life interferon fusion protein and use thereof |
| CN104583240A (en) * | 2012-08-13 | 2015-04-29 | Jw可瑞基因株式会社 | Interferon-[alpha] fusion protein in which cytoplasmic transduction peptide and polyethylene glycol are bonded to one another |
| CN104583240B (en) * | 2012-08-13 | 2017-11-28 | Jw可瑞基因株式会社 | It is combined with the interferon alpha fusion protein of cytoplasm transduction peptide and polyethylene glycol |
| CN102775502A (en) * | 2012-08-16 | 2012-11-14 | 天津禹王生物医药科技有限公司 | Alpha-interferon fusion protein |
| CN106967175A (en) * | 2013-05-15 | 2017-07-21 | 复旦大学 | Long-acting interferon and its production and use |
| CN106729635A (en) * | 2016-12-19 | 2017-05-31 | 武汉市疾病预防控制中心 | A kind of applications of IFN λ 3 in prevention or treatment AIDS-treating medicine is prepared |
| CN116333166A (en) * | 2023-02-08 | 2023-06-27 | 珠海臻谱基因科技有限公司 | Recombinant interferon targeting HIV gp120 protein and application thereof |
| CN116333166B (en) * | 2023-02-08 | 2023-10-03 | 珠海臻谱基因科技有限公司 | Recombinant interferon targeting HIV gp120 protein and application thereof |
| CN117430716A (en) * | 2023-10-16 | 2024-01-23 | 珠海臻谱基因科技有限公司 | Recombinant interferon drug targeting conserved HIV gp41 subunit near-membrane-end outer region and application thereof |
| CN117430716B (en) * | 2023-10-16 | 2024-05-31 | 珠海臻谱基因科技有限公司 | Recombinant interferon drug targeting conserved HIV gp41 subunit near-membrane-end outer region and application thereof |
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| CN101942026B (en) | 2014-08-27 |
| WO2012048653A1 (en) | 2012-04-19 |
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Application publication date: 20110112 Assignee: Chengdu Huiruixinyuan Biotechnology Co., Ltd. Assignor: Chengdu Zenable Bioscience Co., Ltd.|Qiu Junkang|Nie Zimei Contract record no.: 2018510000022 Denomination of invention: Long-acting interferon fusion protein and application thereof Granted publication date: 20140827 License type: Exclusive License Record date: 20180530 |