CN101942026B - 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 studying viral interference phenomenon (virus interference).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 to resistant function.Within 1958, Nagano also observes and has obtained similar result with Kojima.These are all to illustrate in more detail afterwards living organism interferon system 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 obtaining large-scale purification interferon formulation by 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 the cytokine that Interferon, rabbit is produced by one group of induction forms.Interferon, rabbit has wide application, and it can regulate and control to comprise a series of biological functions such as virus multiplication.Interferon, rabbit is divided into two kinds conventionally: I type Interferon, rabbit, also claim viral interferon (Viral IFNs), and comprise 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 generated by virus infection induction.II type Interferon, rabbit is generated by mitogenesis or antigenic stimulation induction.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 are not containing intron (intron), and they are arranged together at people's Chromosome 9 galianconism.Mouse is at its No. 4 karyomit(e) respective regions.Gamma-interferon only has a kind, and it has 3 introns, and it is long-armed that it is positioned at people's No. 12 karyomit(e); Mouse is No. 10 karyomit(e).Although some Interferon, rabbit in body can through some translation after modification glycosylation modified such as N-and O-, main humanα-interferon is not by glycosylation.Alpha-interferon is mainly brought into play its biological effect with monomer, and beta-interferon and gamma-interferon work with homologous dimerization source bodily form formula.Also do not know now why human body has so many alpha-interferon genes.When removing the single beta-interferon gene of mouse by gene knockout (gene knock-out) method, the mouse producing is very responsive to virus infection, this illustrates that numerous alpha-interferon genes 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 to Interferon, rabbit having been carried out to a large amount of successfully fundamental researchs in laboratory, 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 clinical treatment medicine.For alpha-interferon, be used for disease of viral infection the most widely and comprise B-mode and hepatitis C.In addition, alpha-interferon is also 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 relapse of multiple sclerosis.Gamma-interferon is approved for the treatment of chronic heredity immunologic derangement (chronic glomerulus disease).The interferon medicine that You Shuojia biopharmaceutical company production for treating is used.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 being 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.In January calendar year 2001, Peg-Intron-α 2b that U.S. FDA has been ratified the trade(brand)name PEG-Intron of Schering company production is used for the treatment of the chronic hepatitis C patient who did not accept before this interferon therapy.Roche also produces Pegylation and obtains interferon-' alpha ' 2a (commodity are called Pegasys) and be used for the treatment of chronic hepatitis C patient.
Homology Interferon, rabbit (Consensus Interferen, cIFN) is a complete synthesis I type Interferon, rabbit.By analysis, compare 14 kinds of natural alpha-interferon subtype protein matter sequential structures, choose on the corresponding each position of different subtype the amino acid the most often occurring form a new protein sequence (being homologous sequence) thus obtain 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 compared with the two.This may be because cell surface I type Interferon Receptors forms the reason higher compared with alpha-interferon avidity to homology Interferon, rabbit.On the contrary, a little less than homology Interferon, rabbit aspect induction il-1 β generation is compared with alpha-interferon.These results also may reflect the result from the different keying actions of the interactional multiple collaborative albumen of I type Interferon Receptors (accessery proteins).These unique biological characteristicses make 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 the experiment of the various safety of carrying out at a plurality of clinical experiments center and validity, homology Interferon, rabbit and Lymphocytic alpha-interferon [IFN-α (NAMALWA)] treatment having been infected to the chronic hepatitis of high titre hepatitis C virus compares.In the situation that not increasing additional toxicity effect, the validity of homology Interferon, rabbit (18MIU) is obviously better than IFN-α (NAMALWA) (9MIU); Particularly as patient, to have disturbed genotype be 1b when viral that effect is more remarkable.Homology Interferon, rabbit can act on the patient of recurring after treatment chronic hepatitis C patient and the failure of conventional interferon therapy and treatment safely, effectively.Recently homology Interferon, rabbit is also used for the treatment of chronic hepatitis C patient with other anti-liver class chemicals as closed together with ribavirin.Homology Interferon, rabbit has also been successfully used to the treatment of hepatitis B patients.
Yet, interferon therapy is the same with many protein drugs have obvious weak point be exactly transformation period in blood short, alpha-interferon is eliminated rapidly it after being injected into body.Under this short circulating half-life condition, in order to reach result for the treatment of effectively, often adopt multiple-effect amount (every day or inferior on every Wendesdays) long-time medication (6-12 month or longer).In order to improve the pharmacokinetics of alpha-interferon, reduce medicine frequency.Some drugmaker combines the peg molecule of alpha-interferon and-12-KDa or 30-KDa size to have prepared a kind of interferon medicine (Pegglated IFN) of the moulding that narrows.This Peg-Intron molecule can make alpha-interferon extend action time in vivo, has its front clinical therapeutic efficacy.The physical chemistry of Peg-Intron and biological characteristics research have been disclosed to the special antiviral activity of this semi-synthetic interferon molecule obviously relevant to composition and the binding site of the peg molecule of combination.This Pegylation effect can reduce the specific biological specific activity of alpha-interferon.
Albumin (molecular weight is about 67KDa) is rich in protein in blood, and concentration is 50mg/ml (600um).In mankind's transformation period, it is 19 days.Albuminous function comprises and maintains blood plasma pH value, and oncotic pressure, makes to send meta-bolites, lipid acid etc.And it can act on the main pharmaceutical carrier albumen in blood plasma.Human albumin (HSA) long half-lift and stability make it provide a kind of desirable carrier and some fugitive treatment albumen to form fusion molecule.The albumin bound functional zone of staphylococcal protein G and people's complement receptor-1 is formed to fusion rotein can make the latter reach 5 hours 3 times of rat Half-life in vivo increases.Alpha-interferon and Albumin fusion are produced to a kind of brand-new fusion protein molecule.Thereby human genome company is cascaded beta-interferon and HSA encoding gene to give expression to a kind of the two fusion rotein (Albuferon-β). this recombination fusion protein has antiviral and antiproliferation, can start the signal conduction of IFN-stimulated response element (ISRE) mediation.Yet the active specific activity of specific biological of this fusion rotein is still low than alpha-interferon.This may be the space conformation that can affect the latter while merging due to HSA and interferon molecule covalency.
Proved the transformation period that also can extend some fugitive albumen with the non-covalent combination of albumin.But at present not about the correlative study of Interferon, rabbit, and how carrying out real Interferon, rabbit and albumin, to carry out effective non-covalent combination be also unknown at present.
Summary of the invention
The technical problem to be solved in the present invention is can also keep as much as possible its activity in the Half-life in vivo that extends Interferon, rabbit.
The technical scheme that the present invention solves the problems of the technologies described above is to provide a kind of long-acting interferon fusion protein.This long-acting interferon fusion protein is by the C-terminal of Interferon, rabbit, by one section of connection peptides, to connect the affine small peptide of human albumin specificity (HSA-BP) to form.
Preferably, the Interferon, rabbit in above-mentioned long-acting interferon fusion protein is homology Interferon, rabbit.
Wherein, the aminoacid sequence of above-mentioned homology Interferon, rabbit is shown in 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 above-mentioned long-acting interferon fusion protein is shown in SEQ ID NO.2.
H2N-SQRLMEDICLPRWGCLWEDDF-COOH(SEQ ID NO.2)。
Wherein, its aminoacid sequence of above-mentioned long-acting interferon fusion protein is shown in SEQ ID NO.3.
H2N-MCDLPQTHSLGNRRALILLAQMRRISPFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDSSAAWDESLLEKFYTELYQQLNDLEACVIQEVGVEETPLMNVDSIIAVKKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSTNLQERLRRKEGGGSQRLMEDICLPRWGCLWEDDF-COOH(SEQ ID NO.3)。
The aminoacid sequence of the connection peptides that the C-terminal of Interferon, rabbit wherein and human albumin specificity are affine between small peptide HSA-BP is GGG.
The present invention, when above-mentioned long-acting interferon fusion protein is provided, also provides the nucleotide sequence of the above-mentioned long-acting interferon fusion protein of encoding.
Further, the nucleotides sequence of code book invention long-acting interferon fusion protein is classified as shown in SEQ ID NO.4.
ATGTGCGACCTGCCGCAGACCCACTCCCTGGGTAACCGTCGTGCTCTGATCCTGCTGGCTCAGATGCGTCGTATCTCCCCGTTCTCCTGCCTGAAAGACCGTCACGACTTCGGTTTCCCGCAGGAAGAATTCGACGGTAACCAGTTCCAGAAAGCTCAGGCTATCTCCGTTCTGCACGAAATGATCCAGCAGACCTTCAACCTGTTCTCCACCAAAGACTCCTCCGCTGCTTGGGACGAATCCCTGCTGGAAAAATTCTACACCGAACTGTACCAGCAGCTGAACGACCTGGAAGCTTGCGTTATCCAGGAAGTTGGTGTTGAAGAAACCCCGCTGATGAACGTTGACTCCATCCTGGCTGTTAAAAAATACTTCCAGCGTATCACCCTGTACCTGACCGAAAAAAAATACTCCCCGTGCGCTTGGGAAGTTGTTCGTGCTGAAATCATGCGTTCCTTCTCCCTGTCCACCAACCTGCAGGAACGTCTGCGTCGTAAAGAAGGCGGTGGCTCTCAGCGTCTGATGGAAGATATCTGCCTGCCGCGTTGGGGCTGCCTGTGGGAAGATGATTTCTAA(SEQ ID NO.4)。
The present invention also provides the carrier that contains the nucleotide sequence described in claim 6 or 7.
The present invention also provides the host cell that contains the nucleotide sequence described in claim 6 or 7.
Meanwhile, the present invention also provides the purposes of above-mentioned long-acting interferon fusion protein in preparation treatment antiviral or antitumor drug.
The present invention also provides a kind of antiviral or antitumor drug for the treatment of, and this antiviral or antitumor drug be take above-mentioned long-acting interferon fusion protein and as main active ingredient, added pharmaceutically acceptable complementary composition and be prepared from.
Adopt high-throughput screening method (HTS), found that a small peptide (HSA-BP) can optionally carry out high-affinity with human albumin and be combined.The present invention design and the DNA encoding sequence of having synthesized this small peptide, it is passed through to a glycine chain encoding sequence link with the encoding sequence of Interferon, rabbit together with, thereby 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 escherichia coli high-level expression carrier and is gone, can be by escherichia coli high-level expression Interferon, rabbit-HSA-BP (cIFN-HSA-BP) fusion rotein that contains expression vector.Adopt biochemical separation purification method from intestinal bacteria lysate, to obtain highly purified cIFN-HSA-BP fusion rotein.Experiment in vitro proves that this fusion rotein disturbs with homology the identical antiviral and inhibition of cell proliferation effect that have.
Experiment showed, that fusion rotein of the present invention is expelled to interior its transformation period of Mice Body than 12 times of alone cIFN prolongations.This illustrates that cIFN-ABP fusion rotein is in antiviral, the inhibition of cell proliferation biologic activity while with Interferon, rabbit, its transformation period significant prolongation in vivo, thus its clinical treatment effect has obvious advantage than Interferon, rabbit itself.
Accompanying drawing explanation
The gel electrophoresis the result figure of the cIFN-GGG-ABP coding DNA fragment of Fig. 1,0.7KB size.
Fig. 2, gel electrophoresis the result figure.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, 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 the result figure.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 result figure.PBAD-cIFN plasmid after 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).Result demonstration, ECoRV enzyme is cut and can be made pBAD-cIFN plasmid linearization.
The enzyme of Fig. 5, pBAD-cIFNm is cut result Fig. 1: 1kb DNA ladder make (1 μ/10 μ l); 2:pBAD-cIFNm#1 plasmid is cut through ECORV enzyme; 3:pBAD-cIFNm#1 plasmid SacI+Xba double digestion; 4:PCR-Blunt II Topo-cIFN plasmid is through SacI+Xbal double digestion.
The immunoblotting of Fig. 6, production purge process (Western Blot) is identified Fig. 1, the bacterial lysate of not inducing with L-arabinose; 2, the bacterial lysate after L-arabinose induction; 3, cross the sample of Q-Sepharose post; The albumen that 4 final purifications complete.
Fig. 7, Western blot immunoblotting result figure.The 1st road is that restructuring cIFN-ABP can be got off by human albumin antibody co-immunoprecipitation together with human serum albumin, and the 2nd road, for restructuring cIFN, co-immunoprecipitation does not occur.
Fig. 8, restructuring cIFN-ABP fusion rotein are combined test-results with other animal serum albumin.1 human albumin contrasts; 2: dog albumin; 3: rat albumin; 4: monkey albumin; 5: rabbit albumin; 6:: Mouse albumin.
Embodiment
Below in conjunction with accompanying drawing, by concrete enforcement formula, further illustrate but do not limit the present invention.
The acquisition of embodiment mono-restructuring cIFN-ABP protein coding gene and the structure of efficient expression engineering thereof
Artificial chemistry synthesizes cIFN coding gene sequence, and be cloned in pBAD18 plasmid vector (purchased from the handsome Invitrogen of company, Carlsbad, USA, its sequence is referring to J.Bacteriol.177,4121-4130 (1995)), obtain pBAD-cIFN.
The cIFN encoding sequence of first take in this plasmid is template, by PCR method, at its C-end, adds the affine small peptide of human albumin specificity (HSA-BP is called for short ABP) encoding sequence and catenation sequence.PBAD18 plasmid is escherichia coli high-level expression plasmid.CIFN encoding sequence is XbaI and the SacI restriction enzyme site place that is cloned into this plasmid.Therefore, by PCR method, divide the encoding sequence that 2 steps add ABP, connection peptides and restriction enzyme site to remove high efficient expression so that its cIFN-ABP fusion rotein encoding sequence can be cloned in this pBAD18 expression vector again.
PCR reacts I: at cIFN encoding sequence C-terminal, introduce the encoding sequence of connection peptides (GGG) and ABP (SQRLMEDICLPRW GCLWEDDF), in 0.2mlPCR pipe, add following ingredients to mix it:
PCR is reaction time:
I:98 ℃ 2 minutes
II:25 cycle
III:72 ℃ 5 minutes
IV:4 ℃ standby
By reactant electrophoresis in 1% agarose gel.Result is as shown below, as it is consistent to predict the outcome, and has obtained the cIFN-GGG-ABP coding DNA fragment (gel electrophoresis the result is shown in Fig. 1) of a 0.7KB size.
This 0.7kb size is reclaimed to be purified into from agarose gel be used as the template of PCR next time, to introduce XbaI enzyme cutting site for follow-up clone in 3 ' end downstream.
PCR reacts II: in the PCR pipe of 0.2ml, add following composition and mix:
PCR reaction time is with aforementioned PCR reaction time.
Then pBAD-cIFN plasmid is carried out to XbaI+SacI double digestion:
In 37 ℃ of incubations 2 hours.
This plasmid is used to NheI and SphI double digestion in contrast simultaneously:
37 ℃ of incubations 2 hours.
The electrophoresis (gel electrophoresis the result is shown in Fig. 2) in 1% agarose gel by 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 are cut reclaims from agarose gel, purifying.
PCRII reactant is cloned in pCR-BLUNTII ToPo carrier (lnvitrogen, USA) and is gone.
React as follows:
Reactant mixes, and puts 20 ℃ of reactions 10 minutes.
Then reactant is transformed to 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 increasing, preparation plasmid DNA (Qiagen Miniprep Kit) in LB substratum.Plasmid DNA is carried out to double digestion Screening and Identification with XbaI and SacI.Endonuclease reaction is:
37 ℃ of incubations 2 hours.
Endonuclease reaction thing is joined to electrophoresis in 1% agarose gel, result as shown in Figure 3:
By fragment Separation and Recovery purifying from agarose of the SacI-XbaI enzyme of No. 5 colony 0.7kb sizes in Fig. 3.This fragment contains cIFN-GGG-ABP encoding sequence.
Finally, the SacI-XbaI DNA fragmentation that this is contained to cIFN-GGG-ABP encoding sequence is cloned in pBAD18 SacI-XbaI restriction enzyme site and goes.DNA ligation is as follows:
20 ℃ of ligations 4 hours.
As previously mentioned, this DNA ligation reaction is transformed to MachI competence bacterial cell (Invitiogen, USA), transform bacteria is applied on LB+Amp (100 μ g/ μ l) agarose culture plate, measure 37 ℃ and cultivated liquid.Arrange the menu colony inoculation bacterium of increasing next day in 2ml LB+Amp (100 μ g/ml) substratum.Then use Qiagen Mini-Prep kit extracting and purifying bacteria plasmid DNA, plasmid DNA is carried out to enzyme and cut evaluation.First use SacI, XbaI double digestion filters out the positive colony that contains PCRII SacI-XbaI insertion sequence.
By the pBAD plasmid called after pBAD-cIFNm that contains cIFN-ABP insertion sequence filtering out, further pBAD-cIFN and pBAD-cIFNm plasmid are carried out to the comparison of restriction enzyme mapping, in pBAD-cIFN plasmid, only contain an ECoRV restriction enzyme site, therefore, with ECoRV enzyme, cut and can make this plasmid linearization, ECoRV enzyme is cut result as shown in Figure 4.Result demonstration, ECoRV enzyme is cut and can be made pBAD-cIFN plasmid linearization.
Then, choose 1 pBAD-cIFNm plasmid clone and carry out respectively SacI+XbaI double digestion and ECoRV single endonuclease digestion.Method is the same.Owing to itself containing an ECoRV restriction enzyme site except pBAD18 plasmid, so pBAD-cIFNm cuts and can produce 2 DNA fragmentations through ECoRV enzyme, result as shown in Figure 5, completely with predict the outcome consistent.
All results show that pBAD-cIFNm contains correct restriction enzyme site and cIFN-ABP insertion sequence.
Finally, pBAD-cIFNm is carried out to DNA sequencing analysis.Result shows that all cIFN, connection peptides and ABP encoding sequence and implementation sequence meet completely, have reached purpose of design.
High efficient expression and the purifying of embodiment bis-, rcIFN-ABP albumen
TOP10 competence bacterium is purchased from American I nvitrogen company.PBAD-IFNm plasmid is proceeded in TOP10 bacterial cell according to a conventional method, transform bacteria is inoculated in to LB agarose culture plate (containing 100 μ g/ml penbritins) upper, put 37 ℃ of overnight incubation.Get single colony inoculation in 5ml LB liquid nutrient medium (containing 100 μ gml penbritins), put cultivation (225-250RPM) in 37 ℃ of shaking tables and spend the night.Get the 1ml bacterial cultures that spends the night and join in 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.Bacterial cell after results inductions, by bacterial cultures in Sorvall whizzer (RC-5C, Dupont, USA, rotary head model SS-34) in 4 ℃, centrifugal 15 minutes of 6000RPM.With damping fluid (10mM Tris-HCl, pH7.2,10mM EDTA, 100mM NaCl), bacterial precipitation piece is washed once.Then bacterial precipitation thing is suspended in to 100ml Tris damping fluid (50mM Tri-HCl, pH8.5,5mM EDTA, 1mM PMSF).Be placed in ultrasonication bacterium (30 seconds fragmentation/30 second cooling/200-300Wx30 cycles).Bacterial lysate is placed in to 4 ℃ of 12000RPM and within centrifugal 30 minutes, to obtain inclusion body, precipitates piece.Inclusion body is precipitated to piece and be suspended in aforementioned Tris damping fluid (adding 1%deoxycholic acid Septochol), wash 2 times to remove impurity.Then inclusion body throw out is washed once with Tris damping fluid separately.Once again with Milli Q washing finally.
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 20ml damping fluid.Solution is shaken up in room temperature to 5 minutes, then, put centrifugal 15 minutes of 12000RPM to remove insoluble cell impurity.The supernatant of getting containing inclusion body dropwise adds (50mMTris-HCl, pH8.0,1mM EDTA, 2M urea, 10% sucrose, 1mM PMSF, 0.1mM oxidized form and 1mM reduced glutathion (glutamthiones), the stirring of dropping limit, limit in renaturation buffer.Renaturation buffer volume is to add 10 times of inclusion body solution.This solution is placed in to 4 ℃ and stirs 4 hours to allow its albumen beginning renaturation.Measure the pH of solution and be transferred to pH8.4.By this solution for damping fluid (50mMTri s-HCl, pH8.4,1mM EDTA, 2mM urea, 10% sucrose, 1mMPMSF) dialysis.Every dialysis is after 3 hours, then for 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 is put to 4 ℃ of centrifugal 12000RPM 30 minutes, get supernatant through 0.45 μ m membrane filtration.Protein solution after filtering is splined on to Q-Sepharose post (50ml) and carries out purifying.Q-Sepharose previously used damping fluid (50mMTris-HCl, pH8.4,1mM EDTA, 5% sucrose, 1mM PMSF) balance it.Albumen loading speed is 0.5ml/ minute.Then by level pad washing (20 times of column volumes, 1ml/ minute speed) for post.The albumen being combined on post carries out wash-out with 0～1M NaCl salt gradient, and speed is 1ml/ minute.Every pipe 2ml collects, the protein peak of collecting with the monitoring of UV detector.By protein example in SDS-PAGE electrophoretic analysis.And with anti-cIFN interferon antibody, carry out immunoblotting (Western Blot) and identify it (the results are shown in Figure 6).The collection tube that contains cIFN-ABP fusion rotein is merged together, then carries out PBS dialysis (4 ℃, 4 hours * 3). the protein sample after dialysis put-70 ℃ frozen.
The detection of test example one, restructuring cIFN-ABP antiviral activity and with the comparison of cIFN
By 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, cultivate 1 hour.Remove virus, add fresh culture (DMEM+10% foetal calf serum) to continue to cultivate 24 hours, then use Cell Biolabs, the Cytoselect that Inc produces
tMcell Viabilityand cytotoxicity Assay test kit (San Diego, CA, USA) is measured viable cell survival rate.According to interference element dosage one response curve meter, mark EC50 value.
Table 1, antiviral activity and the comparison thereof of restructuring cIFN-ABP in A549 cell
| Interference element | EC50(pg/ml) |
| rc IFN-ABP | 4.8 |
| rcIFN | 4.4 |
| rIFN-α2b | 5.8 |
As can be seen here, restructuring cIFN-ABP is suitable with restructuring cIFN antiviral activity, and the two is all higher than the antiviral activity of restructuring IFN-α 2b.
The combination test of test example two, restructuring cIFN-ABP and human serum albumin
0.5ml human serum albumin (10 μ g/ml) solution (preparing in PBS) is mixed with 0.5ml restructuring cIFN-ABP (10 μ g/ml) or restructuring cIFN (10 μ g/ml) (being all formulated in PBS), put 37 ℃ of incubations 30 minutes, add anti-human albumin antibody (100 μ g/200 μ l) 20 μ l to continue incubation 1 hour.After taking-up, add G albumen-agarose reagent, put room temperature 2 hours (maintenance whipped state).Then centrifugal removal supernatant, washes agarose throw out three times with PBS+0.05%Tween20 damping fluid, finally will in throw out, add 100 μ l 1 * laemmli damping fluids, puts in boiling water 5 minutes.After taking-up, supernatant is joined to 15%SDS-PAGE loading electrophoresis, then carry out Western blot western blot test.With anti-cIFN monoclonal antibody, go to survey the nitrocellulose membrane (antibody application concentration is 1: 1000) after shifting, result as shown in Figure 7.
Test-results shows: restructuring cIFN-ABP can be got off by anti-human albumin antibody co-immunoprecipitation together with human serum albumin, and the two combines to prove them.Restructuring cIFN albumen can not co-precipitation together with human serum albumin, proves between them without combination.
Test example three, restructuring cIFN-ABP fusion rotein are combined test with other animal serum albumin
Mouse, rabbit, monkey, rat, dog serum albumin and agarose is crosslinked.Then agarose-animal white albumen being formulated in to protein content in PBS is 10 μ g/ml.Get the albuminous solution of 0.5ml agarose-animal and 0.5ml restructuring cIFN-ABP (10 μ g/ml) and mix, put 37 ℃ and stir incubations 30 minutes.After taking out, with PBS+0.05%Tween20 damping fluid, wash 3 times.To adding 100 μ l 1x Laemmli damping fluids in throw out, put in boiling water 5 minutes.After taking-up, supernatant is joined to 15%SDS-PAGE loading electrophoresis, then carry out immunoblotting (Westerm blot) test, with anti-cIFN monoclonal antibody, remove to survey the nitrocellulose membrane (antibody application concentration is 1: 1000) after shifting.As shown in Figure 8, restructuring cIFN-ABP's result can and be got off by agarose solids precipitation with all animal white protein binding.
Clearance test in the Mice Body of test example four, restructuring cIFN-ABP
By three groups of female Balb/c mouse (7-8 age in week), 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.After injection, different times is from tail venous blood sampling sample.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 circulation blood sample and the results are shown in Table 2.Result shows that its transformation period is far longer than the common interference element of not transformation.
Clearance test result in table 2 Mice Body
Above-mentioned experiment results proved, fusion rotein of the present invention disturbs with homology the identical antiviral and inhibition of cell proliferation effect that have.And fusion rotein of the present invention is expelled to its transformation period in Mice Body and greatly extends than alone cIFN.This illustrates that cIFN-ABP fusion rotein is in antiviral, the inhibition of cell proliferation biologic activity while with Interferon, rabbit, its transformation period significant prolongation in vivo, thus its clinical treatment effect has obvious advantage than Interferon, rabbit itself.
Claims (6)
1. long-acting interferon fusion protein, it is characterized in that: by the C-terminal of Interferon, rabbit, by connection peptides, connect the affine small peptide HSA-BP of human albumin specificity and form, the aminoacid sequence of the connection peptides that the C-terminal of Interferon, rabbit wherein and human albumin specificity are affine between small peptide HSA-BP is GGG, and the aminoacid sequence of described long-acting interferon fusion protein is shown in SEQ ID NO.3.
Coding long-acting interferon fusion protein claimed in claim 1 nucleotide sequence.
3. the nucleotide sequence of coding long-acting interferon fusion protein according to claim 2, is characterized in that: its sequence is shown in SEQ ID NO.4.
4. the carrier that contains the nucleotide sequence described in claim 2 or 3.
5. the host cell that contains the nucleotide sequence described in claim 2 or 3.
6. long-acting interferon fusion protein claimed in claim 1 is treated the purposes in antiviral in preparation.
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| 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 |
| 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 |
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| Mark S.Dennis et al.Albumin Binding as a General Strategy for Improving the Pharmacokinetics of Proteins.《The Journal of Biological Chemistry》.2002,第277卷(第38期),35035-35043. * |
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