CN101671390A - Human interferon alpha derivatives and preparation and use of pegylated products thereof - Google Patents
Human interferon alpha derivatives and preparation and use of pegylated products thereof Download PDFInfo
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Abstract
The invention relates to human interferon alpha derivatives and preparation and use of pegylated products thereof. The human interferon alpha derivatives are formed by bonding three amino acids to N terminals of human interferon alpha and have a structural formula of R3-R2-R1-interferon, wherein the interferon is the human interferon alpha, and a first position amino acid R1 bonded to the N terminals of the human interferon alpha is a sculpture amino acid or glycine; the second position amino R2 is an aromatic amino acid or glycine; and the third position amino acid R3 is an acidic amino acidor glycine. The invention also provides pegylated derivatives of the human interferon alpha derivatives, a preparation method thereof and use thereof in the preparation of medicaments for treating andpreventing viral infection or lung cancer. The human interferon alpha derivatives have the advantages of high specific activity and high pegylation rate. The pegylated derivatives of the human interferon alpha derivatives have the characteristics of high biological activity and no non-N terminal modified isomers and have the advantages of prolonging in-vivo half-life, reducing plasma clearance and the like.
Description
Technical field
The present invention relates to field of biomedicine technology, be specifically related to the preparation and the purposes of human interferon alpha derivative and pegylated products thereof thereof.
Background technology
Human interferon (interferon, be called for short IFN) be the active protein that exists in the class body with broad-spectrum antiviral, antiproliferative and immunomodulatory function, it is one of cytokine that is used for the earliest clinical treatment, human interferon can be divided into three types, it is human interferon-alpha, human interferon-β, human interferon-gamma also can be further divided into some hypotypes according to the aminoacid sequence difference of various IFN.Prove that through a large amount of clinical studyes human interferon-alpha is a kind of important antitumor and antiviral therapy medicine.At present China clinical most popular mainly be recombinant human interferon-alpha-1a, α-2a and α-1b.
There is more than 20 gene member in the I type family of finder's interferon alpha at present, and wherein most of encode functional protein has about 90% homology each other on nucleotide level.Human interferon-alpha is made up of 165 amino acid, and molecular weight is about 19000 dalton.The clinical therapeutic efficacy of existing human interferon-alpha preparation is still undesirable, and its antiviral specific activity only is 1 * 10
8IU/mg, the bigger shortcoming of side effect when existing heavy dose.
But, no matter be human interferon-alpha-2a, human interferon-alpha-1b and human interferon-alpha-1a, as the protein medicine, because poor stability, the transformation period weak point easily produces antigen antibody reaction etc. in the plasma clearance height, body, is very limited on clinical treatment.Genetic engineering technique makes extensive synthetic human recombination protein become possibility, has solved the immunogenicity problem that heterologous protein causes to a great extent, but still can't overcome shortcomings such as the fast and bioavailability of plasma clearance is low.The result that these shortcomings cause is: need frequent injection human interferon just can reach effective plasma treatment concentration; And, all can cause after the per injection Plasma Concentration than great fluctuation process, form " Feng Yigu " effect of drug level.So just may increase the risk of medical expense and administration inconvenience and untoward reaction.
Polyethyleneglycol modified proteinic technology is a kind of new technology that is used to improve pharmacokinetics character in the protein drug body that grows up over past ten years.It is that activated polyglycol molecule (PEG) is bonded on the protein molecule surface, thereby influence proteinic space structure, finally cause the change of the various biochemical properties of protein, as: chemical stability increases, the ability of opposing protease hydrolysis improves, immunogenicity and toxicity reduce or disappear, and the transformation period prolongs in the body, and plasma clearance reduces or the like.
The PEG component is the amphipathic molecule of the inertia long-chain that is polymerized by the oxygen vinyl monomer.Now existing multiple different PEG molecule can be for utilizing.Its active function gene of the PEG that is activated can be coupled to certain privileged sites (such as amino, sulfydryl or other nucleophiles) of treatment molecule.N-terminal is modified then can obtain the high specific modified outcome, can effectively control the purity of modified outcome, makes the simpler and also easier evaluation of quality product of technology.
Summary of the invention
One of purpose of the present invention provides a kind of new human interferon alpha derivative, is by human interferon-alpha is modified acquisition, and this human interferon alpha derivative has the specific activity height, and antiviral specific activity reaches 5～10 * 10
8IU/mg, stronger 5～10 times than human interferon-alpha, and the high advantage of Pegylation modification rate.
Two of purpose of the present invention provides a kind of carbowax modifier of human interferon alpha derivative, be that human interferon alpha derivative provided by the invention is carried out pointed decoration, obtain the carbowax modifier of the human interferon alpha derivative of high specific, this carbowax modifier biologically active height and the characteristics that do not contain the terminal modified isomer of non-N, and transformation period prolongation in the body, advantages such as plasma clearance reduction.
Three of purpose of the present invention provides the preparation method of the carbowax modifier of above-mentioned human interferon alpha derivative.
Four of purpose of the present invention provides the pharmaceutical applications of human interferon alpha derivative of the present invention and carbowax modifier thereof.
Technical scheme of the present invention is as follows:
Human interferon alpha derivative connects 3 amino acid at the N-terminal of human interferon-alpha, and its structural formula is as follows:
R3-R2-R1-interferon
In the formula, interferon is a human interferon-alpha, and the first amino acids R1 that the N-terminal of human interferon-alpha connects is sulfur-containing amino acid or glycine; The second amino acids R2 is die aromatischen Aminosaeuren or glycine; The 3rd amino acids R3 is acidic amino acid or glycine.
Wherein, described human interferon-alpha comprises human interferon-alpha-2a (IFN α-2a), human interferon-alpha-1a (IFN α-1a), human interferon-alpha-1b (IFN α-1b).In addition, also comprise IFN α-2a, IFN α-1b, three kinds of proteic derivatives of IFN α-1a, analogue, biological activity or medical active fragment.
Wherein, the first amino acids R1 that the N-terminal of human interferon-alpha connects is sulfur-containing amino acid or glycine, and sulfur-containing amino acid comprises methionine(Met), halfcystine, preferred methionine(Met) of R1 or glycine; The second amino acids R2 that N-terminal connects is die aromatischen Aminosaeuren or glycine, and die aromatischen Aminosaeuren comprises phenylalanine, tyrosine, glycine, preferred phenylalanine of R2 or glycine; The 3rd amino acids R3 that N-terminal connects is acidic amino acid or glycine, and acidic amino acid comprises aspartic acid, L-glutamic acid, preferred L-glutamic acid of R3 or glycine.
Wherein, human interferon alpha derivative comprises Glu-Phe-Met-IFN α-2a, Glu-Phe-Met-IFN α-1b, Glu-Phe-Met-IFN α-1a, Gly-Gly-Gly-IFN α-2a, Gly-Gly-Gly-IFN α-1b, Gly-Gly-Gly-IFN α-1a, Glu-Tyr-Met-IFN α-2a, Glu-Tyr-Met-IFN α-1b, Glu-Tyr-Met-IFN α-1a, Glu-Gly-Met-IFN α-2a, Glu-Gly-Met-IFN α-1b or Glu-Gly-Met-IFN α-1a.
The present invention also provides the nucleotide sequence of the above-mentioned human interferon alpha derivative of encoding, and this nucleotide sequence formula has the nucleotide modification sequence at the N-terminal of human interferon-alpha, this modification sequence can encode successively above-mentioned R3, R2 and the described amino acid of R1.The preparation of above-mentioned nucleotide sequence can be by gene recombination technology or synthetic acquisition of artificial full gene.As the cDNA sequence (SEQ IDNO:2) that is used for making up the Yeast expression carrier contain Glu-Phe-Met-IFN α-2a (IFN-D) among the embodiment 1 is to obtain through full gene is synthetic.
The present invention also provides the expression vector of the nucleotide sequence that contains the above-mentioned human interferon alpha derivative of encoding.Preferred expression vector is a Yeast expression carrier, also can select other conventional expression vector.The structure of this expression vector can insert construction of expression vector in the carrier with the purpose fragment by conventional molecule clone technology.This expression vector can be in expressing engineering bacteria effective expression, can access above-mentioned human interferon alpha derivative through separation and purification.
The present invention also provides the pegylated products thereof of above-mentioned human interferon alpha derivative, is to connect activated polyglycol molecule (PEG) by amido linkage on the amino of the N-terminal of described human interferon alpha derivative, and its structural formula is as follows:
Wherein, polyoxyethylene glycol has straight chain or branched structure, and molecular-weight average is 10000-40000 dalton.
Prepare the method for the pegylated products thereof of above-mentioned human interferon alpha derivative, may further comprise the steps:
(a) utilize the engineering bacteria that contains expression vector to prepare human interferon alpha derivative solution;
(b) polyoxyethylene glycol and human interferon alpha derivative linked reaction;
(c) purifying of polyoxyethylene glycol human interferon alpha derivative.
The present invention also provides above-mentioned human interferon alpha derivative and the purposes of carbowax modifier in the medicine of preparation treatment or prevention viral infection or tumor disease thereof, above-mentioned human interferon alpha derivative and carbowax modifier thereof can be mixed with radiotherapy and/or the chemotherapy of liquid preparation combined utilization in malignant tumour with medically acceptable carrier, also can be used for the treatment of or prevent viral infection.Those skilled in the art can give the Pegylation human interferon alpha derivative by dosage 0.5～3 μ g/kg by injection according to practical situation.
The present invention is under the constant situation of the original aminoacid sequence of human interferon-alpha, connects 3 amino acid at the N-terminal of peptide molecule, forms human interferon alpha derivative.Human interferon alpha derivative after reorganization has the specific activity height, and antiviral specific activity reaches 5～10 * 10
8IU/mg, stronger 5～10 times than human interferon-alpha, and the high advantage of Pegylation modification rate.
The contriver finds through sequential analysis: the 1st N-terminal of the α-1a of human interferon-alpha family, α-2a and α-1b is cysteine residues, its-SH and 29 Cys-disulfide linkage formed between the SH, thereby the peptide molecule that makes human interferon-alpha becomes curling sterie configuration, if such polypeptide N end residue is modified with PEG, its modification rate is very low, only reaches 2～5%.Through gene recombination technology methionine(Met) or glycine on the N of human interferon-alpha termination, and linear die aromatischen Aminosaeuren or glycine in the 2nd connection of its extension successively, acidic amino acid or glycine in the 3rd connection.Because of die aromatischen Aminosaeuren has bigger sterically hinderedly, stoped terminal 3 amino acid to curl, thereby fully exposed free-NH of N end to intramolecularly
2, help free-NH of the N end of the human interferon-alpha after PEG derives to reorganization
2Modify, finish PEG N-terminal is carried out pointed decoration, obtain the polyethyleneglycol derivative of the human interferon alpha derivative of high specific.This deriving except that the proteic general advantage of PEGization such as having such as stable, good water solubility, antigenicity be low, especially biologically active is high and do not contain the characteristics of the terminal modified isomer of non-N, and transformation period prolongation in the body, advantages such as plasma clearance reduction, and lay a solid foundation for later clinical application.
The invention provides new human interferon alpha derivative and polyoxyethylene glycol human interferon alpha derivative thereof,, can treat or prevent viral infection or tumor disease for scientific research and clinical treatment provide new drug molecule.
Description of drawings
Fig. 1 is that IFN-D Bijie Yeast expression carrier makes up synoptic diagram among the embodiment 1.
Fig. 2 is the SDS-PAGE detected result of IFN-D among the embodiment 2.Among the figure 1, Marker:SDS-PAGE lower molecular weight standard protein; 2, cross CM post target compound; 3, cross DEAE post target compound; 4, fermented liquid enriched material.
Fig. 3 is IFN-D SDS-PAGE detected result after ALD-PEG20KD modifies among the embodiment 3.Among the figure 1, Marker:SDS-PAGE lower molecular weight standard protein; 2, mPEG (20KD)-IFN-D target compound; 3, the sample of IFN-D after ALD-PEG20KD modifies; 4, IFN-D sample.
Embodiment
Describe the present invention by the following examples in detail.Related in the following embodiments experiment material all can buy or pass through preparation method's acquisition of this area routine if no special instructions by market.
The secreting, expressing of embodiment 1 Glu-Phe-Met-IFN α-2a in methanol yeast
1, goal gene obtains and design
The aminoacid sequence of Glu-Phe-Met-IFN α-2a (hereinafter referred to as IFN-D) is shown in SEQ IDNO:1, and sequence length is 168 amino acid.
Make corresponding codon into the yeast preferences know the CDNA of IFN α-2a by Genebank after.And in N-terminal adding Glu-Phe-Met corresponding nucleotide sequence.This CDNA sequence is used for Bijie zymic PIC9K expression plasmid and makes up.The expression plasmid that makes up is realized secreting, expressing after transforming GS115 host bacterium.Therefore in design, add KEX
2Enzyme recognition site CTC GAG AAA AGA, wherein CTC GAG is the XhoI restriction enzyme site.Two terminator codon TGA TAA of 3 ' end introducing simultaneously and Not I enzyme are cut sequence GCG GCCGC.SEQ ID NO:2 is the cDNA sequence that is used to make up the Yeast expression carrier of IFN-D.Described IFN-D cDNA sequence is to entrust the Shanghai total man worker of biotechnology company limited gene synthetic.This sequence flush end after checking order inserts in the pUC57 plasmid called after pUC-IFN-D.
2, the structure of two of expression plasmid pPIC9-IFN-D, pPIC9K-IFN-D kinds of different carriers.
Goal gene among the pUC57-IFN-D is reclaimed the purpose fragment with XhoI and NotI endonuclease double digestion glue, in order to connecting.
Expression vector pPIC9 plasmid is reclaimed big fragment as carrier with endonuclease XhoI and NotI double digestion, be connected with the T4 ligase enzyme with above-mentioned IFN-D fragment, 16 ℃, 30min uses CaCl again
2Method is transformed among the E.coli DH5 α, uses the enzyme cutting method screening positive clone, called after pPIC9-IFN-D.
Reclaiming big fragment as carrier behind expression vector pPIC9K plasmid usefulness Sal I and the Sac I double digestion, reclaim small segment as goal gene after again the pPIC9-IFN-D plasmid being used Sal I and Sac I double digestion, be connected with the T4 ligase enzyme with above-mentioned pPIC9K people's fragment, use CaCl
2Method is transformed among the EcoliDH5 α, uses the enzyme cutting method screening positive clone, called after pPIC9K-IFN-D.
3, express the acquisition of engineering bacteria
The pPIC9K-IFN-D plasmid with linearization of Sac I after among the methanol yeast GS115 and the chosen process of positive recombinant be: (1) preparation competence GS115; (2) the plasmid pPIC9K-IFN-D of linearization of adding; (3) join behind the mixing in the conversion cup; (4) electric shock conversion condition: 0.5kv, 2.5Uf, 1500hm; (5) use 1.0ml, the 1.0M sorbyl alcohol washes out; (6) coat on the MD/His flat board, 30 ℃, grow single bacterium colony after 24 hours; (7) picking list bacterium colony separate application selects substratum (MD) or methyl alcohol to select on the substratum (MM) in glucose, and growth is mut rapidly on the MM flat board
+, other be that mutsc methyl alcohol slowly utilizes type; (8) renewed vaccination is to the flat board that contains aminoglycosides antibiotics G418 different concns, and the high single bacterium colony of screening number of copies is preserved standby.
4, the expression of pPIC9K-IFN-D engineering bacteria
Picking mono-clonal engineering bacteria shakes in the bottle at 500ml in 25ml BMGY and cultivates on the MD flat board, and 30 ℃, the 300rpm overnight incubation.OD
600During=4.0-6.0, with aseptic centrifuge tube 3000rpm, 4 ℃ of centrifugal supernatants that go, with 25ml BMM substratum suspension thalline, change over to and contain abduction delivering in the 250ml BMM nutrient solution, added 100% methyl alcohol in per 24 hours, final concentration is 0.5%, 30 ℃, and 300rpm cultivated 96 hours.Cleer and peaceful precipitation in the centrifugal reservation of fermented liquid.Through electrophoresis and HPLC analysis expression of results be: reorganization Glu-Phe-Met-IFN α-2a expression amount in supernatant liquor reaches 50mg/L.
The purifying of embodiment 2IFN-D
1, positively charged ion gel column (as CM Sepharose F.F. gel) chromatography:
Adopt pH 3.8～4.6 acetate buffers to carry out upper prop, wash-out, target compound is collected in the electrophoresis monitoring.Use pH7.5～8.5 Tris-HCl buffered soln that target compound is dialysed then.
2, negatively charged ion gel column (as DEAE Sepharose F.F. gel) chromatography:
Adopt pH7.5～8.5Tris-HCl buffered soln to carry out upper prop, wash-out, collect target compound.With pH7.5～8.5 phosphate buffer solns target compound is dialysed again.
3, SDS-PAGE detects: get above-mentioned CM post and DEAE post pass liquid and target peak detects, test-results shows that fermented liquid passes through above column chromatography purification, obtained purity and reached IFN-D more than 95%.The results are shown in shown in the accompanying drawing 2.
The preparation and the purifying process of sample modified in embodiment 3PEG coupling:
1, the IFN-D sample is dialysed with phosphate buffered saline buffer, add equimolar ALD-PEG 20KD then and modify at 2～15 ℃, the reaction times is 24～36 hours.The modification sample that obtains is carried out SDS-PAGE detect, test-results shows that after the PEG coupling was modified, molecular weight improved, and brings up to nearly 40000 dalton by 19000 original dalton, and the modification rate reaches more than 40%, obtains target compound mPEG (20KD)-IFN-D.The result as shown in Figure 3.
2, the purifying of mPEG (20KD)-IFN-D positively charged ion gel column (as SP Sepharose F.F. gel):
To modify sample, use to regulate specific conductivity be 4.0～5.0 with termination reaction, and last SP post carries out purifying.Use acetate buffer to carry out wash-out, collect target compound.With phosphate buffer soln target compound is dialysed, get final product.
Adopt the extracorporeal antivirus effect activity of cytopathic-effect inhibition assay by WISH-VSV systems measurement human interferon, be method well-known in the art, concrete with reference to three appendix XC of 2005 editions Pharmacopoeias of the People's Republic of China " biological activity determination of human interferon ".
Present embodiment has been measured the extracorporeal antivirus effect activity of human interferon alpha derivative IFN-D and polyoxyethylene glycol human interferon alpha derivative mPEG (20KD)-IFN-D, and its extracorporeal antivirus effect determination of activity the results are shown in Table 1 (more than be the mean value of three measurement results).
The extracorporeal antivirus effect activity of table 1 IFN α-2a, IFN-D and mPEG (20KD)-IFN-D
Experiment conclusion:
Human interferon alpha derivative after reorganization has the specific activity height, and antiviral specific activity reaches 5 * 10
8IU/mg is stronger 5 times than human interferon-alpha.Active decline after Pegylation is modified, active reservation 20%.
Embodiment 5, usefulness ELISA method are surveyed the Plasma Concentration of polyoxyethylene glycol human interferon alpha derivative mPEG (20KD) in the mouse body-IFN-D sample
1, experiment purpose:
Plasma Concentration in the survey mouse body after mPEG (20KD)-IFN-D, the metabolism of IFN-D sample different number of days.
2, experimental design and experimental technique:
Get 98 of Kunming mouses, female male half and half, body weight is divided into high and low two dosage groups at random about 22-25g, and 42 every group, 14 of blank groups.The high dose group dosage is that mPEG (20KD)-IFN-D 50 micrograms/kg and low dose group dosage are IFN-D 10 micrograms/kg, the sample that low dose group is 10 micrograms/kg in the first day abdominal injection concentration of testing, and every day is by this dosed administration, administration 7 days.MPEG (20KD)-IFN-D sample that high dose group is 50 micrograms/kg in only first day abdominal injection concentration of experiment.Two administration experimental group were respectively taken a blood sample six from the mouse eye at the 1st, 2,3,4,5,6,7 day that tests, and adopted blank group blood sample simultaneously and did blank.2000 rev/mins centrifugal 5 minutes, get supernatant liquor, survey the content of sample in the blood plasma according to the specification sheets on the ELISA test kit.(y=1.3229Ln (x)-0.0449, R2=0.9637), absorbancy substitution equation is per sample obtained concentration value again to obtain logarithmic equation according to the absorbance of standard pipe.
3, experimental result: see Table 2.
Table 2 10 μ g/kg low dosage administration groups and 50 μ g/kg high dosage administration group Plasma Concentrations are relatively
4, experiment conclusion:
After testing the Plasma Concentration numerical value comparative descriptions after mPEG (20KD) in the mouse body-IFN-D, the metabolism of IFN-D sample different number of days the human interferon alpha derivative pegylated products thereof can extension body in the transformation period.
More than the preparation method and the purposes of human interferon alpha derivative provided by the present invention and pegylated products thereof thereof have been done detailed introduction.It is to be noted; the described content of embodiment be for better implement the present invention preferred embodiment; protection scope of the present invention is not limited to the described technical scheme of above-mentioned embodiment; and should be as the criterion with the described flesh and blood of claims, any possible change that those skilled in the art did is only otherwise the scope that the flesh and blood that breaks away from claim of the present invention all belongs to the present invention to be protected.
Sequence table
＜110〉1, Hainan Sihuan Cardiovascular ﹠ Cerebrovascular Medicines Research Institute Company Limited; 2, Beijing Sihuan Pharmaceutical Co., Ltd; 3, Hainan Sihuan Pharmaceutical Co., Ltd
＜120〉preparation of human interferon alpha derivative and pegylated products thereof thereof and purposes
<130>MP081195
<160>2
<170>PatentIn?version?3.3
<210>1
<211>168
<212>PRT
<213>Homo?sapiens
<400>1
Glu?Phe?Met?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Ser?Arg?Arg
1 5 10 15
Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg?Lys?Ile?Ser?Leu?Phe?Ser?Cys
20 25 30
Leu?Lys?Asp?Arg?His?Asp?Phe?Gly?Phe?Pro?Gln?Glu?Glu?Phe?Gly?Asn
35 40 45
Gln?Phe?Gln?Lys?Ala?Glu?Thr?Ile?Pro?Val?Leu?His?Glu?Met?Ile?Gln
50 55 60
Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser?Ala?Ala?Trp?Asp
65 70 75 80
Glu?Thr?Leu?Leu?Asp?Lys?Phe?Cys?Thr?Glu?Leu?Tyr?Gln?Gln?Leu?Asn
85 90 95
Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly?Val?Gly?Val?Thr?Glu?Thr?Pro
100 105 110
Leu?Met?Lys?Glu?Asp?Ser?Ile?Leu?Ala?Val?Arg?Lys?Tyr?Phe?Gln?Arg
115 120 125
Ile?Thr?Leu?Tyr?Leu?Lys?Glu?Lys?Lys?Tyr?Ser?Pro?Cys?Ala?Trp?Glu
130 135 140
Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser?Phe?Ser?Leu?Ser?Thr?Asn?Leu
145 150 155 160
Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu
165
<210>2
<211>504
<212>DNA
<213>Homo?sapiens
<400>2
gaattcatgt?gtgatttgcc?tcaaactcat?tctttgggtt?ctagaagaac?tttgatgttg 60
ttggctcaaa?tgagaagaat?ttctttgttt?tcttgtttga?aggatagaca?tgattttggt 120
tttcctcaag?aagaatttgg?taaccaattt?caaaaggctg?aaactattcc?tgttttgcat 180
gaaatgattc?aacaaatttt?taacttgttt?tctactaagg?attcttctgc?tgcttgggat 240
gaaactttgt?tggataagtt?ttacactgaa?ttgtaccaac?aattgaacga?tttggaagct 300
tgtgttattc?aaggtgttgg?tgttactgaa?actcctttga?tgaaggaaga?ttctattttg 360
gctgttagaa?agtactttca?aagaattact?ttgtacttga?aggaaaagaa?gtactctcct 420
tgtgcttggg?aagttgttag?agctgaaatt?atgagatctt?tttctttgtc?tactaacttg 480
caagaatctt?tgagatctaa?ggaa 504
Claims (16)
1, human interferon alpha derivative is characterized in that, connects 3 amino acid at the N-terminal of human interferon-alpha, and its structural formula is as follows:
R3-R2-R1-interferon
In the formula, interferon is a human interferon-alpha, and the first amino acids R1 that the N-terminal of human interferon-alpha connects is sulfur-containing amino acid or glycine; The second amino acids R2 is die aromatischen Aminosaeuren or glycine; The 3rd amino acids R 3 is acidic amino acid or glycine.
2, human interferon alpha derivative according to claim 1, it is characterized in that human interferon-alpha comprises human interferon-alpha-2a, human interferon-alpha-1a, human interferon-alpha-1b or described three kinds of proteic derivatives, analogue, biological activity or medical active fragment.
3, human interferon alpha derivative according to claim 1 is characterized in that, described sulfur-containing amino acid comprises methionine(Met), halfcystine.
4, human interferon alpha derivative according to claim 1 is characterized in that, described die aromatischen Aminosaeuren comprises phenylalanine, tyrosine.
5, human interferon alpha derivative according to claim 1 is characterized in that, described acidic amino acid comprises aspartic acid, L-glutamic acid.
6, human interferon alpha derivative according to claim 1 is characterized in that, R1 is methionine(Met) or glycine.
7, human interferon alpha derivative according to claim 1 is characterized in that, R2 is phenylalanine or glycine.
8, human interferon alpha derivative according to claim 1 is characterized in that, R3 is L-glutamic acid or glycine.
9, human interferon alpha derivative according to claim 1, it is characterized in that human interferon alpha derivative comprises Glu-Phe-Met-IFN α-2a, Glu-Phe-Met-IFN α-1b, Glu-Phe-Met-IFN α-1a, Gly-Gly-Gly-IFN α-2a, Gly-Gly-Gly-IFN α-1b, Gly-Gly-Gly-IFN α-1a, Glu-Tyr-Met-IFN α-2a, Glu-Tyr-Met-IFN α-1b, Glu-Tyr-Met-IFN α-1a, Glu-Gly-Met-IFN α-2a, Glu-Gly-Met-IFN α-1b or Glu-Gly-Met-IFN α-1a.
10, the nucleotide sequence of each described human interferon alpha derivative of coding claim 1-9.
11, the expression vector that contains the described nucleotide sequence of claim 10.
12, expression vector according to claim 11, described expression vector is a Yeast expression carrier.
13, the pegylated products thereof of each described human interferon alpha derivative of claim 1-9, it is characterized in that, connect activated polyglycol molecule (PEG) by amido linkage on the amino of the N-terminal of described human interferon alpha derivative, its structural formula is as follows:
14, pegylated products thereof according to claim 13, wherein peg molecule has straight chain or branched structure, and molecular-weight average is 10000-40000 dalton.
15, the method for preparing the described pegylated products thereof of claim 13 is characterized in that may further comprise the steps:
(a) utilize the engineering bacteria that contains expression vector to prepare human interferon alpha derivative solution;
(b) polyoxyethylene glycol and human interferon alpha derivative linked reaction;
(c) purifying of polyoxyethylene glycol human interferon alpha derivative.
16, described human interferon alpha derivative of claim 1 and carbowax modifier thereof the purposes in the medicine of preparation treatment or prevention viral infection or tumor disease.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8143214B2 (en) | 2007-08-16 | 2012-03-27 | Pharmaessentia Corp. | Protein-polymer conjugates |
| CN106868035A (en) * | 2017-03-13 | 2017-06-20 | 上海赛伦生物技术股份有限公司 | A kind of preparation method of restructuring horse Interferon alpha 1 |
| CN108721603A (en) * | 2017-04-14 | 2018-11-02 | 中国医学科学院基础医学研究所 | The purposes of Alfacon-1 a in the preparation of medicament for cancer treatment |
| US11559567B2 (en) | 2014-11-06 | 2023-01-24 | Pharmaessentia Corporation | Dosage regimen for pegylated interferon |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5595732A (en) * | 1991-03-25 | 1997-01-21 | Hoffmann-La Roche Inc. | Polyethylene-protein conjugates |
| CN1475505A (en) * | 2002-08-13 | 2004-02-18 | 北京英莱特生物技术开发有限公司 | Human composite type alpha interferon protein N terminal tag structure |
-
2008
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8143214B2 (en) | 2007-08-16 | 2012-03-27 | Pharmaessentia Corp. | Protein-polymer conjugates |
| US11559567B2 (en) | 2014-11-06 | 2023-01-24 | Pharmaessentia Corporation | Dosage regimen for pegylated interferon |
| CN106868035A (en) * | 2017-03-13 | 2017-06-20 | 上海赛伦生物技术股份有限公司 | A kind of preparation method of restructuring horse Interferon alpha 1 |
| CN106868035B (en) * | 2017-03-13 | 2020-06-05 | 上海赛伦生物技术股份有限公司 | Preparation method of recombinant equine Interferon-alpha-1 |
| CN108721603A (en) * | 2017-04-14 | 2018-11-02 | 中国医学科学院基础医学研究所 | The purposes of Alfacon-1 a in the preparation of medicament for cancer treatment |
| CN108721603B (en) * | 2017-04-14 | 2020-11-13 | 中国医学科学院基础医学研究所 | Use of interferon alpha-1 a for the preparation of a medicament for the treatment of cancer |
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Effective date of registration: 20220325 Address after: 101114 Beijing city Tongzhou District Zhangjiawan Town Qi Shanzhuang East Village Patentee after: BEIJING SIHUAN PHARMACEUTICAL Co.,Ltd. Patentee after: Hainan four ring Pharmaceutical Co., Ltd. Address before: 570216 a06-2, Haikou Free Trade Zone, Hainan Province Patentee before: Hainan Sihuan Cardiovascular Drug Research Institute Co.,Ltd. Patentee before: BEIJING SIHUAN PHARMACEUTICAL Co.,Ltd. Patentee before: HAINAN SIHUAN PHARMACEUTICAL Co.,Ltd. |