CN101967196A - Interferon fusion protein, preparation thereof and application thereof - Google Patents
Interferon fusion protein, preparation thereof and application thereof Download PDFInfo
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Abstract
The invention relates to an interferon fusion protein, preparation thereof and application thereof. The interferon fusion protein is a fusion protein formed by connecting an Fc fragment of the human immunoglobulin G and the human interferon, or connecting the human interferon and the Fc fragment of the human immunoglobulin through a connecting peptide. The preparation of the fusion protein comprises the following steps of: preparing an interferon gene and an Fc fragment gene of the human immunoglobulin G respectively, connecting the interferon gene and the Fc fragment gene and then constructing a recombinant expression vector which contains the connecting fragment; and transforming host cells by using the recombinant expression vector, culturing the host cells, recovering the recombinant fusion protein from cell culturing substances and purifying the recovered product to obtain the recombinant fusion protein. The fusion protein in the invention can be applied in the preparation of antivirus medicaments, antitumor medicaments and immune adjusting medicaments.
Description
Technical field
The present invention relates to recombinant human immunoglobulin (Ig) FC (crystalizable fragment, Fc) preparation of fused interferon, Zhi Bei Interferon, rabbit can obviously prolong Interferon, rabbit action time in vivo in this way, can be used for the treatment of human relative disease.
Background technology
The Fc structural domain of antibody comprises at least two CH structural domains of each chain usually, and its dimerization forms the Fc structural domain.The Fc structural domain is responsible for providing the antibody mediated effect function, comprises decision antibody half life and distribution complement-fixing in vivo and in conjunction with the ability of cell surface Fc acceptor.The character of Fc structural domain makes it be called useful therapeutical agent.Many researchs have been blended in the Fc structural domain other non-antibody protein, receptor protein for example, and Yi Naxipu for example also can be with Fc structural domain syzygy with the reagent that makes a search, and it helps proteic detection and purifying the Fc label.
The human IgG immunoglobulin (Ig) is intravital main antibody, and it the intravital transformation period is about 20 days the people, and its stability is because the Fc fragment of IgG can combine with newborn Fc acceptor (FcRn), avoids IgG to enter in the lysosome and is degraded.Therefore, the Fc fragment of IgG is used to connect and compose fusion rotein with activated protein, with the transformation period in the body that improves activated protein, reaches long lasting purpose.IgG can play the cell killing effect by the complement binding site activating complement on the Fc fragment.According to the length of hinge area and the difference of Fc fragment aminoacid sequence, human IgG is divided into 4 hypotypes (γ 1, γ 2, γ 3 and γ 4), and wherein IgG Fc γ 2 has lower inducing cytotoxic effect, and IgG Fc γ 1 is the strongest.The building mode of IgG fusion rotein is that the segmental N end of the Fc of IgG (Hinge-CH2-CH3) fragment or CH (CH1-Hinge-CH2-CH3) is linked to each other with the C end of activated protein mostly, with the influence of avoiding construction of fusion protein to cause the activated protein biological activity.In addition, if activated protein is brought into play its biological activity with the form of homodimer, the intermolecular disulfide bond that the cysteine of Fc γ fragment and hinge area constitutes can strengthen and the dimeric formation of stable fusion rotein.Human leukocyte function antigen 3 (LFA-3) for example, Tumor Necrosis Factor Receptors II (TNFRII), interleukin 12s (IL-12) etc., these fusion roteins obviously prolong the transformation period of cytokine in experimentation on animals when keeping the cytokine biological function.In addition, the fusion rotein of IgG can obtain the high efficient and convenient purifying by Protein A affinity chromatography.Although therefore a lot of cytokines and the fusion rotein of IgG also are in the preliminary stage of research, owing to its high efficiency, long half time and purifying easily characteristics caused concern widely.
Interferon, rabbit (IFN) is a kind of multifunctional cytokine, and its at first found biological activity is to resist the resistance virus infection.For many years, the antiviral activity of Interferon, rabbit is its unique biological function.Nowadays, find to disturb to have many other biological functions, as the growth, differentiation and the immunomodulatory isoreactivity that act on cell may have bigger biological significance.Interference have two diverse families, is divided into I type and II type.I type Interferon, rabbit mainly comprises IFN α and IFN β, and II type Interferon, rabbit is IFN γ.The main effect of I type Interferon, rabbit is antiviral, and IFN α and IFN β use a kind of acceptor jointly, but have diverse immunogenicity.IFN α is mainly produced by human leukocyte, is called LeIF, and IFN β is mainly produced by inoblast, is called fiblaferon.IFN α has a lot of hypotypes, as IFN α-1, IFN α-2, IFN α-3 etc. 20 kinds of hypotypes is arranged approximately, also has hypotype in addition, and as IFN α-1a, IFN α-1b, IFN α-2a, IFN α-2b etc., IFN α has tens kinds more than.IFN β still finds no hypotype.II type Interferon, rabbit is mainly produced by the T cell; Main activity is to participate in immunomodulatory, is important immune-regulating factor in the body, claims type II interferon again.IFN γ has only a kind of protein of activity form, does not have other hypotype.Interferon, rabbit is the adjusting protein with extensive biologic activity, has antiviral, immunoregulatory effect, kinds of tumor cells had complementaryly kill or suppress proliferation function, stop or the process of normal cell that slow down to tumour cell transformation thereby can also suppress oncogene expression.Since beginning to develop recombinant human interferon alpha 2 the eighties, more than 60 state approval listing arranged at present.Be widely used in hepatitis B, third liver; Leukemia; Bleb, condyloma; Kidney, bladder cancer; Multiple virus disease and tumor treatment such as lymphoma, myelomatosis, melanoma.
But there is some not ideal enough part in common Interferon, rabbit, because the interferon molecule amount is little, easily by glomerular filtration, so the subcutaneous injection post-absorption is very fast, the transformation period is short, easily by enzymic hydrolysis etc., generally will inject every day or inject at least weekly three times, it be convenient inadequately to use.And during diseases such as interferon therapy hepatitis, tumour, similar rheumatism and multiple sclerosis, treatment cycle usually reaches several months even several years, and the frequent medication of this long-term heavy dose has increased patient's misery and medical expense and easily produced toxic side effect such as fever, tired, appetite stimulator and influenza-like symptom.
The Interferon, rabbit that polyoxyethylene glycol (PEG) is modified is the longest long-acting interferon of transformation period that uses clinically of FDA approval, PEG is connected with Interferon, rabbit can prolong the Interferon, rabbit transformation period in the blood, make interferon activity can extend to a week, an intramuscular injection once weekly, be equivalent to common Interferon, rabbit intramuscular injection every day effect, and can improve curative effect.But the IFN price that PEG modifies is expensive, the modification process complexity of PEG and Interferon, rabbit, thus make polyethyleneglycol modified Interferon, rabbit cost too high, increased patient's medical expense.In the fusion rotein patent of the serum albumin of BIO ENGINEERING INST MILITARY and Interferon, rabbit (publication number CN140518A), serum albumin be provided with connection peptides (Gly during interferon protein is connected
4Ser) n, n are the integer of 1-10.The sequence that includes foreign protein in fusion rotein, the existence of foreign protein sequence still lacks documents and materials to the influence of its immunogenicity and result of treatment.
In view of Interferon, rabbit short and lower characteristics of long-acting interferon activity at present of transformation period in vivo, the present invention has designed a kind of New-type long-acting Interferon, rabbit, is mainly used in antivirally, improves aspects such as immunizing power and oncotherapy.
Summary of the invention
In order to overcome the short and present lower deficiency of long-acting interferon activity of transformation period in the existing Interferon, rabbit body, the invention provides a kind of interferon fusion protein and application thereof, this interferon fusion protein can stimulate body to the immunne response of virus infection, long half time in vivo, the therapeutic action that plays maximum and the potential side effect that reduces traditional Interferon, rabbit, has solved the problem that prior art exists effectively.
The technical solution adopted in the present invention is:
A kind of interferon fusion protein is the fusion rotein that the Fc fragment by immunoglobulin G while links to each other and constitutes with human interferon, or the fusion rotein that connects and composes of the Fc fragment of people's Interferon, rabbit by a connection peptides and human normal immunoglobulin.
The above-mentioned Interferon, rabbit of saying can comprise any member of Interferon, rabbit family.In a specific examples, the n cell factor that Interferon, rabbit is produced by the cell of virus infection, such Interferon, rabbit is at " peptide growth factor and receptor II thereof " (Sporn and Roberts, Spring-Verlag Heidelberg, New York Inc., USA.PP3-38) in by Vilcek(1991) be called " Interferon, rabbit ", this title is used till today.
The special example of Interferon, rabbit comprises, but be not limited to Interferon, rabbit IFN-ɑ-1(IFNA-1), IFN-a-2(IFNA-2), IFN-a-4(IFNA-4), IFN-a-5(IFNA-5), IFN-a-6(IFNA-6), IFN-a-7(IFNA-7), IFN-a-8(IFNA-8), IFN-a-10(IFNA-10), IFN-a-12(IFNA-12), IFN-a-13(IFNA-13), IFN-a-14(IFNA-14), IFN-a-16(IFNA-16), IFN-a-17(IFNA-17), IFN-a-21(IFNA-21), IFN--1 (IFNB-1) IFN--2(IFNB-2, IL-6), IFN-γ-1 (IL-29), IFN-γ-2 (IL-28A) and IFN-ε etc.
SeqID No.2,4,6, the 8th, the concrete several aminoacid sequences that belong to interferon fusion protein of the present invention, the part in the said interferon fusion protein of the present invention and SeqID No.2,4,6,8 have the sequence of at least 90% homology.
Interferon, rabbit can directly be connected in IgG Fc albumen, and (N-terminal of hinge hinge area+CH2+CH3) or C-terminal form interferon analogue, also can connect Fc and Interferon, rabbit to form any albumen by connection peptides, as Interferon, rabbit-connection peptides-Fc, or Fc-connection peptides-Interferon, rabbit.Connection peptides length is 2-100 amino acid preferably, is more preferably 5-50 amino acid, most preferably is 14-30 amino acid.Connection peptides length can be as short as Fc the steric hindrance that Interferon, rabbit forms is kept minimum, for example (G
4S)
3-4Connection peptides helps Interferon, rabbit and its receptors bind.
The said interferon fusion protein of the present invention prepares by the following method:
(1) make the Fc fragment gene of interferon gene and immunoglobulin G respectively,
(2) connect interferon gene and Fc gene;
(3) make up the recombinant expression vector that contains above-mentioned (2) junction fragment;
(4) with the recombinant expression vector transformed host cell that obtains in above-mentioned (3),
(5) cultivate host cell, recovery and purifying obtain recombination fusion protein (being interferon fusion protein) from cell culture then.
In a preferred embodiment, the invention provides a kind of recombinant eukaryon expression vector pFc-IFN that comprises Fc-IFN fusion gene of the present invention, and the host cell pFc-IFN/CHO that contains this recombinant expression vector.
Step (5) is use the recombinant expression vector transformed host cells being suitable under the condition of this fusion rotein cultivating, and from cell culture the step of recovery and this recombination fusion protein of purifying.In a preferred embodiment, host cell is through cell cultures, centrifugal after, supernatant liquor is used affinity chromatography and sieve chromatography purifying respectively.In a preferred embodiment, affinity chromatography is a ProteinA sepharose FF affinity column, and molecular sieve is Superdex 200 PREP grad posts.
In addition, the invention still further relates to a kind of mammalian cell and produce the method for the high efficiency, low cost of interferon fusion protein, particularly the lactation kinetocyte produce IgG Fc respectively with IFN-a1b, IFN-a2a, IFN-a2b, IFN-a-8(IFNA-8), the fusion rotein formed of IFN-γ-1 (IL-29), IFN-γ-2 (IL-28A) and IFN-ε.Long half time in the very high and blood plasma of the stability of above-mentioned any fusion rotein in blood plasma, it is external, and intravital biological protection function is identical with traditional Interferon, rabbit.
The present invention also provides a kind of interferon fusion protein to be used to prepare the application of medicine.These application comprise the pharmaceutical preparation be made up of above-mentioned interferon fusion protein and the effective dose of treatment, and can be that any pharmacy is acceptable can make the stable any vehicle form of interferon analogue with pharmaceutical preparation.The human normal immunoglobulin that pharmaceutical preparation can further make natural or reorganization with or different interferon analogues.
The present invention further provides and contain the pharmaceutical composition of interferon fusion protein of the present invention (IFN-Fc fusion rotein) as activeconstituents and pharmaceutically acceptable carrier, and the purposes of this pharmaceutical composition in the medicine of treatment relative disease, comprise fields such as antiviral, oncotherapy and immunomodulatory.
The Fc-IFN fusion rotein of mentioning herein has the aminoacid sequence of Fc-IFN fusion rotein, but the replacement that wherein one or more amino-acid residues have been guarded by different amino-acid residues, and resulting polypeptide can be used for implementing the present invention.It is known in the art that conserved amino acid replaces.Cause such replacement principle to comprise, described replacement rule such as MD by Dayholf.In particular, the conserved amino acid replacement occurs in the amino acid family that is associated with its acidity, polarity or side chain size.
The method of producing and operating polynucleotide molecule disclosed herein is well known by persons skilled in the art, and can finish according to the recombinant technology of describing.The expression vector that can be used for expressing IFN-Fc fusion rotein sequence of the present invention is known in the art, comprising the recombinant phage dna that contains the specific coding sequence, plasmid DNA and cosmid DNA expression vector, the typical prokaryotic expression plasmid that can contain polynucleotide molecule of the present invention through processing comprises PUC8, PUC9, PBR322 and PBR329, the pET serial carrier, PQE50 or the like, the typical carrier for expression of eukaryon that can contain polynucleotide molecule of the present invention through processing comprises moulting hormone induction type mammalian expression system, based on the expression system of bringing up the cell virus promoter-enhancer with based on baculovirus expression system.
Can be used for implementing host cell of the present invention can be eucaryon or prokaryotic cell prokaryocyte.Such host cell comprises and singly is not limited to microorganism, for example use recombinant phage dna, the bacterium that plasmid DNA or cosmid DNA carrier transform, the perhaps yeast that transforms with recombinant vectors, or zooblast, as insect cell with recombinant viral vector such as baculovirus infection, or with the mammalian cell of recombinant viral vector such as adenovirus or vaccinia virus infection etc.For example, can use coli strain, as the BL21 bacterial strain.Eukaryotic cell comprises yeast cell, but also can effectively utilize mouse, mammalian cell such as hamster, ox, monkey or human cell line.The eukaryotic host cell that can be used for expressing recombinant protein of the present invention comprises CHO, and HEK293, NIH-3T3 cell etc.
The pharmaceutical composition that contains recombinant fusion protein of the present invention can be used for the treatment of relative disease, especially in fields such as antiviral, oncotherapy and immunomodulatorys.
It will be understood by those skilled in the art that pharmaceutical composition of the present invention is applicable to various administering modes, oral administration for example, percutaneous dosing, intravenously administrable, intramuscular administration, topical, nose administration etc.According to the administering mode that is adopted, recombinant protein medicine composition of the present invention can be made various suitable formulations, wherein comprise the polypeptide of the present invention and at least a pharmaceutically acceptable pharmaceutical carrier of at least a effective dose.
The example of appropriate dosage forms is a tablet, capsule, and sugar coated tablet, granula, oral liquid and syrup, the ointment and the medicine that are used for skin surface paste aerosol, nasal spray, and the sterile solution that can be used for injecting.
The pharmaceutical composition that contains recombinant protein of the present invention can be made solution or lyophilized powder to be used for parenteral admin, section's adding appropriate solvent or other pharmaceutically useful carriers reconfigure powder before use, liquid formulations generally is damping fluid, isotonic solution and the aqueous solution.
Also comprise in the formulation by other conventional components, as sanitas, stablizer, tensio-active agent, damping fluid, the salt of adjusting osmotic pressure, emulsifying agent, sweetener, tinting material, seasonings etc.The stablizer optimization citric acid sodium that drug regimen species of the present invention use, glycine, N.F,USP MANNITOL, ganglioside etc.The injection liquid drugs injection or the preferred prescription of freeze-dried powder that contain pharmaceutical composition of the present invention comprise, Fc-IFN, NaCl 4mg, vitriol 3.02mg, glycine 10mg or N.F,USP MANNITOL 15mg, water for injection 0.5ml.
The consumption of pharmaceutical composition peptide species of the present invention can another in a big way in the change, those skilled in the art can be according to some factors always as the kind according to disease, the degree that is in a bad way, patient body weight, formulation, factors such as route of administration are determined easily.
Advantage of the present invention:
1) when molecule number is identical, compares, have similar biologic activity with natural IFN;
2) demonstrate the transformation period and the stability of significant prolongation in for experiment at the medicine of medicine;
3) the Fc fragment of selecting immunoglobulin (Ig) is avoided the generation of side effect as merging fragment;
4) expression amount height, good stability is easy to amplify and produces, and cost is low.
After 100 generations of engineering cell strain continuous passage of the present invention, still can express the Fc-IFN fusion rotein by stably excreting, cultivate, with the expression amount in the ELISA method detection supernatant by the suspension culture mode, add up data inferior surplus in the of 50, expression amount is all at 500-700mg/L.Aspect purifying, because fusion rotein content height in cells and supernatant, and the affinity chromatography step in the purifying process has very high purification efficiency, every milliliter of gel can be easy to amplify and produce in conjunction with the albumen of 50mg.
Description of drawings
Fig. 1: the gene splicing flow process that shows signal peptide-Fc-IFN ɑ 2a fusion gene.
Fig. 2: show pcr amplification signal peptide-Fc-IFN ɑ 2a fusion gene.M:15000bp DNA ladder; 1: the product of primer pFc1 and pIFN2 amplification; 2: the product of primer p 3 and pIFN2 amplification; 3: the product of primer p2 and pIFN2 amplification; 3: the product of primer p1 and p IFN2 amplification.
Fig. 3: the enzyme of recombinant plasmid pIFN ɑ 2a-Fc is cut qualification result (1% agrose electrophoresis).Wherein swimming lane M is a molecular weight standard, 1-6: the double digestion of six strain clones of picking at random.
Fig. 4: the rProtein A Sepharose Fast Flow elution profile that shows Fc-IFN ɑ 2a.
Fig. 5: show the expression product after Fig. 5: SDS-PAGE detects purifying.M: standard molecular weight; 1: reduced form SDS-PAGE; 2: non-reduced type SDS-PAGE.
Fig. 6: IFN ɑ 2a and Fc-IFN ɑ 2a fusion rotein are to the protection test of virus attack cell.
Fig. 7: IFN ɑ 2a and the Fc-IFNA2a fusion rotein stability in the time of 37 ℃.
Fig. 8: IFN ɑ 2a and the stability of Fc-IFN ɑ 2a fusion rotein in the time of 50 ℃.
Fig. 9: IFN ɑ 2a and Fc-IFN ɑ 2a fusion rotein were at 72 hours long-acting.
Figure 10: IFN ɑ 2a and Fc-IFN ɑ 2a fusion rotein were at 16 days long-acting.
Embodiment
SEQ ID NO:1 is a fusion rotein IgG2 Fc-IFN ɑ 2a nucleotide sequence;
SEQ ID NO:2 is the aminoacid sequence of encoding fusion protein IgG2 Fc-IFN ɑ 2a;
The nucleotide sequence of Seq ID No.3 coding Immunoglobulin IgG2 Fc and people IFNa2b fusion rotein;
The aminoacid sequence of Seq ID No.4 coding Immunoglobulin IgG2 Fc and people IFNa2b fusion rotein;
The nucleotide sequence of Seq ID No.5 coding Immunoglobulin IgG2 Fc and people IFNa1b fusion rotein;
The aminoacid sequence of Seq ID No.6 coding Immunoglobulin IgG2 Fc and people IFNa1b fusion rotein;
The nucleotide sequence of Seq ID No.7 coding Immunoglobulin IgG2 Fc and people IFNa-8 fusion rotein;
The aminoacid sequence of Seq ID No.8 coding Immunoglobulin IgG2 Fc and people IFNa-8 fusion rotein.
Embodiment 1:IFN ɑ 2a
Goal gene and human IgG2 Fc(hinge region+CH2+CH3) acquisition of fragment gene
According to IFN ɑ 2a gene order (NM_000605), deliver gene Synesis Company and synthesize.With the total RNA of normal people's lymphocyte is template, primer is PFc1:5 ' GAC AAG ACC CAC ACC TGT C 3 ', PFc2:5 ' GCCGCCAGATCCGCCTCCGCCCTTTCCGGGGCTCAG 3 ', RT-PCR amplification IgG2 Fc fragment, reaction process is as follows, reverse transcription reaction: 70 ℃ 10 minutes, 42 ℃ of 94 ℃ of 4 ℃ of coolings in 5 minutes in 30 seconds.PCR reaction: 94 ℃ of sex change 30 seconds; Annealed 30 seconds for 55 ℃; 72 ℃ were extended 1 minute, and reacted 30 circulations.72 ℃ were extended 5 minutes more then.After reaction was finished, 1% agarose gel electrophoresis detected the RT-PCR product.The schema of whole gene splicing as shown in Figure 1, Fig. 2 represents that we have obtained estimating the dna fragmentation (about 1350bp) of size.
Embodiment 2: construction of recombinant plasmid
1) overlapping extension splicing (over-lap) connects IFN ɑ 2a and Fc gene: Primers design is as follows: according to IFN ɑ 2a and human IgG2 Fc (hinge region+ CH2+ CH3) the following primer of cDNA sequences Design:
PFc1:?5’?GAC?AAG?ACC?CAC?ACC?TGT?C?3’
PFc2:?5’?GCCGCCAGATCCGCCTCCGCCCTTTCCGGGGCTCAG?3’
PIFN1:5’TCTGGCGGCGGAGGAAGTGGCGGAGGCTCTGGCTGCGACCTGCCTCAG?3’
PIFN2:?5’?CGCGGATCCTCA?CTC?TTT?GGA?CTT?CAG?3’
PIFN2 contains the BamH1 restriction enzyme site.Contain glycine-Serine connection peptides (Gly3Ser) sequence among PFc2 and the PIFN1
2) PCR reaction system
In 50 μ l PCR reaction tubess, set up following reaction system
| 10× |
5?μl |
| cDNA | 2?μl(10?ng) |
| PFc1?(10 mM) | 2?μl |
| PFc2:?(10 mM) | 2?μl |
| dNTP | 4?μl |
| The Taq enzyme | 0.5μl(2.5U) |
| The |
3?μl |
Mend H2O to 50 μ l,
Reaction conditions is as follows:
94℃ 3?min
94℃ 45?sec
55℃ 30?sec
72℃ 1?min
72℃ 7?min
Be cooled to 4 ℃,
30 Cycles after reaction finishes, carry out evaluation of 1.0% Agarose electrophoresis and rubber tapping and reclaim the purpose band.
Second takes turns amplification IFN, sets up following reaction system in 50 μ l PCR reaction tubess
| 10× |
5? |
| IFN | |
| 2?μl(10?ng) | |
| PIFN1(10 mM) | 2?μl |
| PIFN2(10 mM) | 2?μl |
| dNTP | 4?μl |
| The Taq enzyme | 0.5μl(2.5U) |
| The |
3?μl |
Mend H2O to 50 μ l
94℃ 3?min
94℃ 30?sec
55℃ 30?sec
72℃ 1?min
72℃ 7?min
Be cooled to 4 ℃,
30 Cycles after reaction finishes, carry out evaluation of 1.0% Agarose electrophoresis and rubber tapping and reclaim the purpose band.
Third round OVER-LAP pcr amplification Fc-IFN sets up following reaction system in 50 μ l PCR reaction tubess
| 10× |
5?μl |
| The first, two takes turns |
2?μl(10?ng) |
| PFc1?(10 mM) | 2?μl |
| PIFN2(10 mM) | 2?μl |
| dNTP | 4?μl |
| The Tag enzyme | 0.5μl(2.5U) |
| The |
3?μl |
Mend H2O to 50 μ l
94℃ 3?min
94℃ 30?sec
55℃ 30?sec
72℃ 1?min
72℃ 7?min
Be cooled to 4 ℃,
30 Cycles after reaction finishes, carry out evaluation of 1.0% Agarose electrophoresis and rubber tapping and reclaim the purpose band.
3) primer extension composite signal peptide sequence
P1?5’-CCCAAGCTTGCCACCATGGGCTGGTCCTGCATCATCCTGTTTCTGG-3’
P2?5'-ATCATCCTGTTTCTGGTGGCTACCGCCACCGGAGT-3’
P3?5’-CTACCGCCACCGGAGTGCATTCTGACAAGACCCACACCTGTC-3’
In 50 μ l PCR reaction tubess, set up following reaction system
| 10× |
5?μl |
| Fc- |
2?μl(10?ng) |
| P1?(10?mM) | 2?μl |
| PIFN2?(10 mM) | 2?μl |
| dNTP | 4?μl |
| The Taq enzyme | 0.5μl(2.5U) |
| The |
3?μl |
Mend H2O to 50 μ l,
Reaction conditions is as follows:
94℃ 3?min
94℃ 45?sec
55℃ 30?sec
72℃ 1?min
72℃ 7?min
Be cooled to 4 ℃,
30 Cycles after reaction finishes, carry out evaluation of 1.0% Agarose electrophoresis and rubber tapping and reclaim the purpose band.
In 50 μ l PCR reaction tubess, set up following reaction system
| 10× |
5?μl |
| First |
2?μl(10?ng) |
| P2?(10?mM) | 2?μl |
| PIFN2?(10 mM) | 2?μl |
| dNTP | 4?μl |
| The Tag enzyme | 0.5μl(2.5U) |
| The |
3?μl |
Mend H2O to 50 μ l,
Reaction conditions is as follows:
94℃ 3?min
94℃ 45?sec
55℃ 30?sec
72℃ 1?min
72℃ 7?min
Be cooled to 4 ℃,
30 Cycles after reaction finishes, carry out evaluation of 1.0% Agarose electrophoresis and rubber tapping and reclaim the purpose band.
In 50 μ l PCR reaction tubess, set up following reaction system
| 10× |
5?μl |
| Second takes turns |
2?μl(10?ng) |
| P1?(10?mM) | 2?μl |
| PIFN2?(10 mM) | 2?μl |
| dNTP | 4?μl |
| The Tag enzyme | 0.5μl(2.5U) |
| The |
3?μl |
Mend H2O to 50 μ l,
Reaction conditions is as follows:
94℃ 3?min
94℃ 45?sec
55℃ 30?sec
72℃ 1?min
72℃ 7?min
Be cooled to 4 ℃,
30 Cycles after reaction finishes, carry out evaluation of 1.0% Agarose electrophoresis and rubber tapping and reclaim the purpose band.
The PCR product is cut glue and is reclaimed the purpose fragment after electrophoretic separation, sets up following endonuclease reaction system subsequently respectively:
| The Fc- |
16?μl(1μg) |
| 10×Buffer? |
2?μl |
| Hind? |
1 μl(15U) |
| BamH?I | 1?μl(15U) |
Mend H2O to 20 μ l, 37 ℃ of reaction 8 h.Carrier pYD7 adopts same enzyme to cut the system enzyme and cuts.
5) enzyme is cut product 1% agarose electrophoresis, and rubber tapping is reclaimed, and system connects: s
| 10×Ligation Buffer? | 2?μl |
| The Fc-IFN endonuclease bamhi | 15?μl(0.2 pmol) |
| The pYD7 linearized |
2?μl(0.03 pmol) |
| T4? |
1?μl(350U) |
Mend H
2O to 20 μ l, 16 ℃ of reaction 12 h.
6) flat board is drawn in the DH5 α inoculation of the frozen preservation of glycerine, be inverted overnight incubation for 37 ℃;
7) the picking mono-clonal is to the test tube that 3 ml LB are housed, 37 ℃ of 220 rpm jolting 12 h;
8) draw in 1 ml bacterium liquid to the 1.5 ml centrifuge tube, 4 ℃ of centrifugal 3 min of 12000g abandon
Clearly;
9) with the resuspended bacterial sediment of CaCl2 of 400 μ l 0.1mol/l precoolings, centrifugal 3 min of 12000g,
Abandon supernatant; With the resuspended once more bacterial sediment of CaCl2 of 200 μ l 0.1mol/l precoolings, put on ice and spend the night;
10) will connect liquid 20 μ l and all add in the 200 μ l competence bacteriums, put 1h on ice;
11) 42 ℃ of heat-shocked 90 sec put 5 min in the ice rapidly; The LB nutrient solution that adds 37 ℃ of preheatings of 800 μ l;
12) 37 ℃, 220 rpm joltings, 1 h all coats the LB that contains 50 μ g/ml Kan after centrifugal
Flat board is inverted overnight incubation for 37 ℃.
13) at random 4 mono-clonals of picking to the test tube of the 3ml LB nutrient solution that contains 30 μ g/ml AMP, 37 ℃ of 220 rpm jolting 12 h, after bacterium colony PCR identifies, extracting plasmid (being undertaken) 1.0% Agarose electrophoresis detection purity and roughly quantitative by OMEGA plasmid a small amount of extraction agent box specification sheets;
14)
BamHI and
NdeI carries out double digestion, and 1.0% Agarose electrophoresis is identified.
15) recombinant plasmid is delivered to the order-checking of Invitrogen company, obtained the pYD7 of correct sequence and the recombinant plasmid called after pFc-IFN ɑ 2a of Fc-IFN α 2.
Fig. 3 represents that enzyme cuts the result of evaluation, shows that we have obtained positive colony.Complete fused protein Fc-IFN α 2a gene and aminoacid sequence such as SEQ ID NO:1 and SEQ ID NO:2.Other sequences can finally obtain following nucleotide sequence with similar method and be respectively SeqID N0:3,5,7, and corresponding aminoacid sequence is respectively SeqID No. 4,6,8.
Embodiment 3:CHO stably express strain screening and evaluation
1) gets pFc-IFN ɑ 2a plasmid among the embodiment 2 of a large amount of preparations of 10ug, utilize liposome Lipofectin2000 transfection CHO cell.Ratio in 1:5 goes down to posterity two days later, adds the G418 screening of 0.4mg/ml, and visible clone formed in 10 days.Digest the mono-clonal that 168 edges obviously separate, cell state is good at random and be inoculated in 7 24 orifice plates cultivations (first round screening).
2) get in the cultivation after three days and please detect the Expression of Fusion Protein situation with ELISA, therefrom select 45 clones of expression male and be inoculated in 2 24 orifice plates (second takes turns screening) and 16 orifice plate (be used for protecting and plant) respectively, cultivate after 4 days, get supernatant and detect the Expression of Fusion Protein situation, therefrom choose 6 higher clone: A2-6, B1-5, C6-3, D5-5, D6-4, D6-5 of expression and further screen with limiting dilution assay with ELISA.
3) inoculate (5 cells/well/200ul) (the third round screening) of 96 orifice plates respectively, treat that cell covers with back (after about 13 days), go culture supernatant to detect the Expression of Fusion Protein situation with ELISA, B4-3, A6-4, C1-5 are the clone of homogeneous, picking is expressed 6 higher clone's inoculation 24 orifice plates, each parallel 2 hole respectively.After treating that cell covers with, wherein a hole is used for protecting kind, and 6 orifice plates are inoculated in another hole.After treating that the interior cell of 6 orifice plates covers with, extract genomic dna and total RNA, identify the segmental situation that exists of genomic insertion that is integrated into PCR, insert segmental transcribing (promptly expressing) situation with the RT-PCR evaluation, B4-3, A6-4, C1-5 are correct clone.
4) get B4-3, A6-4 respectively, C1-5 inoculates (1 cells/well/200ul) (the four-wheel screening) of 96 orifice plates, treat that cell covers with back (after about 15 days), get to cultivate and please detect the Expression of Fusion Protein situation with ELISA, three clones are the clone of homogeneous, with wherein expressing the highest amplification respectively, protecting and plant, carry out next step expression and purifying.After pFc-IFN ɑ 2a transforms Chinese hamster ovary celI, with the cell called after CHO-FI cell of stably express.
Embodiment 4: preparation Fc-IFN ɑ 2a fusion rotein
The CHO-FI cell of large scale culturing transfection, supernatant liquor is regulated pH to 7.3 with the NaOH of 1M after centrifugal, adsorb in order to 10mM PB (pH7.3) equilibrated rProteinA-Sepharose F.F. (Parmacia) affinity column, again with same damping fluid washing affinity column to the OD value of 280nm less than 0.01.Citrate buffer solution with 0.05～0.1M washes fusion rotein, collects elution peak and uses 200mM Na immediately
2HPO
4Regulate PH to 7.0.After the sample concentration, last molecular sieve Superdex 200 prep grade posts are used 20 mM PBS in advance, and (contain 150mMNaCL, pH7.2) damping fluid balance is with sample purifying on the sample after concentrating.Affinity chromatography figure is Fig. 4, and the protein electrophoresis collection of illustrative plates behind the purifying is seen Fig. 5, and purity can reach more than 98%.
Embodiment 5: preparation is the injection/freeze-dried of active ingredient with Fc-IFN ɑ 2a
Prescription:
Fc-IFN ɑ 2a100mg
NaCL 8g
Na
2HPO
4·12H
2O 5.16g
NaH
2PO
4·2H
2O 0.874g
Glycine 15g
N.F,USP MANNITOL 30g
Water for injection 1000ml
PH value 7.2
Be distributed into injection water injection or freeze-drying that 0.5ml/ props up after the sterile filtration, make freeze-dried powder.
Embodiment 6:Fc-IFN ɑ 2a fusion rotein is to the protection test of virus attack cell
The antiviral activity of Fc-IFN ɑ 2a and fusion rotein thereof induces the cytopathy degree that causes to determine by cytokine protection human fetal amnion WISH cell opposing stomatitis herpesvirus (VSV).(4.5 * 105 cells/ml) place 96 holes (100ul/ hole) to WISH cell in advance; with the IFN ɑ 2a of 3 times of gradient dilutions or Fc-IFN ɑ 2a fusion rotein 37 ℃ of following incubations 18 hours; the absorption value of measuring the cell of violet staining in elisa plate can be determined the activity of WISH cell; in this analysis method; IFN ɑ 2a is when median effective dose (ED50) 0.45 ± 0.04pm; it is 50% that its protection VSV induces the ability of the WISH cell pathology that causes; and interferon fusion protein has the antiviral 50%(Fig. 6 of natural interferon when median effective dose (ED50) 0.2 ± 0.3Pm concentration).Because Fc-IFN ɑ 2a is bivalent molecule, but estimation result as calculating according to volumetric molar concentration, the specific activity of Fc-IFN ɑ 2a fusion rotein is 2 times of IFN ɑ 2a.
Example 7: use the enzyme immunoassay interferon analogue
A kind of ordinary method when application compares mensuration with the active of IFNa2a and Fc-IFNa2a in the enzyme immunoassay mensuration unknown sample and with the active interferon standard substance of known organism, Interferon, rabbit-ɑ ELISA test kit that Chemicon company produces is a double antibody sandwich method.Chemikine
TMThe goat anti-rabbit antibodies plate is caught special Interferon, rabbit-a complex body, and the Interferon, rabbit that contains in the complex body-a antibody combines with Interferon, rabbit a, the competitive part of biological link coupled Interferon, rabbit-a(), sample or standard substance competition Interferon, rabbit-a specific antibody binding site.Therefore when Interferon, rabbit in the sample-a concentration increases, reduced by the link coupled Interferon, rabbit a of antibody capture amount, but with streptavidin alkaline phosphatase link coupled substrate reactions analysis quantitative analysis, the content of Interferon, rabbit-a is compared with making with the standard substance curve in the sample, can calculate Interferon, rabbit-a biological activity and the content in the unknown sample.And determine the biologic activity of reorganization Fc-IFNa2a fusion rotein in contrast with the people recombinant interferon-a of isolating human interferon-a, expressing cho cell in the urine.
The result shows that Fc-IFNa2a has identical biological activity with IFNa2a.
Embodiment 8: measure interferon analogue in the active stability of external biological
With Fc-IFN ɑ 2a is example, to Fc-IFN ɑ 2a under 37 ℃ and 50 ℃ of conditions, carrying out stability in different time points measures, get 50U (0.5ng) by the people IFNa2a of bacterial expression or the people Fc-IFNa2a fusion rotein of 50U (19.6ng) purifying, place and contain 200ul, do not contain in the RPMI1640 nutrient solution of serum and other components, test tube is sealed mouth and is placed water-bath, take out sample in different time points, place-80 ℃ of preservations at once, after treating that whole sample collections are good, carry out biological activity determination with the virus infection WISH cell, and set up contrast by standard operating procedure.Result such as Fig. 7 show, IFN ɑ 2a is all active 37 ℃ of forfeitures in following 24 hours, but at 37 ℃ after 24 hours, the biological activity of Fc-IFN ɑ 2a is without any variation, even its antiviral activity still preserved bioactive 3/4 after 20 days.Under 50 ℃ of conditions, IFN-a-2a lost all active after 24 hours, but after 6 days, the biological activity of interferon analogue still keeps original active 90%, result such as Fig. 7, Fig. 8 show the prolongation of interferon analogue shelf time, and envrionment temperature is had better resistance.
Embodiment 9: the retention time of interferon analogue in blood plasma
Measured the retention time of Fc-IFNa2a and Interferon, rabbit IFNa2a in animal plasma.15ng(about 1 * 10
3U) people IFN-a-2a adds IgG Fc or the 60g(about 1 * 10 of 45ng from expressing cho cell
3U) people IFNa2a analogue is diluted to the 100ul injection of solution respectively and goes in the mouse body, gathers blood sample (50ul) respectively at different time then.The last day of experiment, all mouse of being tried are all gathered blood sample 500ul, and adding EDTA is centrifugal together in miniature centrifuge tube, and supernatant liquor places-80 ℃ of storages.Chemkine with Chemicon company
TMThe human interferon radioimmunological kit test all pick up from injected IFNa2a(contrast).The serum sample of interferon analogue animal.The result shows that Fc-IFNa2a keeps the longer time that is not degraded (Fig. 9 and 10) than exposed IFNa2a in blood plasma.Injecting the existence that still can detect Fc-IFNa2a in 16 days, this result has 14-20 days plasma half-life consistent with immunoglobulin Fc.The plasma sample of injection Fc-IFNa2a still can detect the antiviral activity of protection WISH cell at the 16th day, the result shows and do not protect cell to avoid the function of virus attack in the reference substance.This long lasting biological function gives fusion rotein new clinical application.
Claims (6)
1. interferon fusion protein is characterized in that it being the fusion rotein that the Interferon, rabbit by the Fc fragment of immunoglobulin G while and people links to each other and constitutes, or the fusion rotein that connects and composes of the Fc fragment of people's Interferon, rabbit by a connection peptides and human normal immunoglobulin.
2. interferon fusion protein according to claim 1 is characterized in that wherein said Interferon, rabbit is selected from IFN-a-1, IFN-a-2, IFN-a-4, IFN-a-5, IFN-a-6, IFN-a-7, IFN-a-8, IFN-a-10, IFN-a-12, IFN-a-13, IFN-a-14, IFN-a-16, IFN-a-17, IFN-a-21, IFN--1, IFN--2, IFN-γ-1, IFN-γ-2 and IFN-ε.
3. according to the described interferon fusion protein of claim 1, it is characterized in that wherein said connection peptides is (G
4S)
3-10
4. the preparation method of an interferon fusion protein may further comprise the steps:
(1) make the Fc fragment gene of interferon gene and immunoglobulin G respectively,
(2) connect interferon gene and Fc gene;
(3) make up the recombinant expression vector that contains above-mentioned (2) junction fragment;
(4) with the recombinant expression vector transformed host cell that obtains in above-mentioned (3),
(5) cultivate host cell, recovery and purifying obtain recombination fusion protein from cell culture then.
5. a pharmaceutical composition is characterized in that, is made up of fusion rotein and pharmaceutically acceptable carrier as the claim 1 of activeconstituents.
6. the application of the described fusion rotein of claim 1 in preparing antiviral, antitumor and immunoregulation druge.
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| CN103087200B (en) * | 2013-01-28 | 2014-08-27 | 江苏众红生物工程创药研究院有限公司 | Pig IFN (interferon) gamma-Fc fusion protein as well as coding gene and expression method of pig IFN (interferon) gamma-Fc fusion protein |
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| CN105669871A (en) * | 2016-04-19 | 2016-06-15 | 中国药科大学 | Fusion protein of thymulin alpha1 |
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