CN103044554B - Human urinary trypsin inhibitor (hUTI) of reorganization-dimerization and preparation method and application thereof - Google Patents
Human urinary trypsin inhibitor (hUTI) of reorganization-dimerization and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a human urinary trypsin inhibitor (hUTI) of reorganization-dimerization and preparation method and application thereof, wherein the hUTI protein of reorganization-dimerization successively comprises amino acid residue sequence of human UTI, a peptide linker and a human IgGFc variant from N-terminal to C-terminal. The hUTI protein of reorganization-dimerization has the in-vitro biological activity similar with that of the human urinary trypsin inhibitor, higher biological activity in vivo and an extended half-life period in vivo.
Description
Technical field
The present invention relates to molecular biology and medical field.More specifically, the present invention relates to a kind of have Recombinant Human Urinary trypsin inhibitor dimer similar or more high biological activity, its preparation method and application with natural human urinary trypsin inhibitor.
Background technology
Human urine trypsin inhibitor (human Urinary Trypsin Inhibitor, hUTI), also referred to as ulinastatin or UTI, is the glycoprotein of the proteinase inhibitor class that contains two Kunitz structural domains.HUTI is present in normal people's urine and blood, and molecular weight is about 40kD.HUTI has multiple physiologically active, and as the restraining effect to serine stretch protein enzyme family, wherein serine stretch protein enzyme family comprises trypsinase, alpha-chymotrypsin, plasminogen, kethepsin-G and leukocyte elastase.HUTI also has immunoregulation effect, can lower the release of pro-inflammatory cytokine, such as tumor necrosis factor-alpha (TNF-α), il-1 (IL-1) and interleukin (IL-6) (referring to Park etc., J Korean Med Sci, 25:128-34,2010; Sato etc., Jpn J Thorac Cardiovasc Surg, 48:428-34,2000; Park etc., Korean J Anesthesiol, 58:334-37,2010; Inoue etc., Expert Opin Investig Drugs, 19:513-20 2010; Yang etc., Biologicals 38:552-6,2010; Pugia etc., Clin Chem Lab Med43:1-16,2005; Pugia etc., Adv Clin Chem 44:223-245,2007).In addition, during hUTI also passes through and PDGF-D (PDGF-DD)/the active dimer of PDGF-BBR disturbs the signal path of its mediation, and then the transfer of inhibition malignant mesothe cell, by the propagation of inhibition tumor cell and the expression of lowering CXCL4 and MMP-9 albumen, can significantly reduce mammary cancer and transplant the growth of the breast tumor in nude mice model (referring to Yaguchi etc., Cancer Lett 288:214-18,2010; Sun etc., J Int Med Res 38:967-76,2010; Kobayashi etc., Biol Chem 384:749-754,2003), prompting hUTI has potential use aspect oncotherapy.In addition, further studies confirm that hUTI can suppress the hydrolytic activity of trypsinase and other proteolytic enzyme in Various Tissues, and bring into play part or whole body anti-inflammatory action (referring to Bae etc., Inflammation35:176-82,2012; Takano etc., Lab Invest 89:833-9,2009; Inoue etc., J Clin Biochem Nutr43:139-142,2008; Ueki etc., J Biosci Bioeng 104:315-20,2007; Molor-Erdene etc., Thromb Haemost 94:136-45,2005; Hirose etc., Biol Pharm Bull 21:651-56,1998; Wu etc., Hepatobiliary Pancreat Dis Int 8:53-8,2009).To implementing the high-risk patient with operation body internal jugular vein injection hUTI of general anesthesia, can improve blood microcirculation, amelioration of inflammation reaction.In addition, hUTI also can be used for the treatment of various acute inflammation, comprise that acute pancreatitis, systemic inflammatory responses syndromes, acute circulatory failure, disseminated inravascular coagulation (DIC) and multiple organ dysfunction syndrome are (referring to Jong In Han, Korean J Anesthesiol, 58:325-327,2010).
Research of Animal Model for Study shows, the shock reaction of hUTI induced by endotoxin mediation and hepatic ischemia-reperfusion injury have provide protection (referring to Inoue etc., Expert Opin Investig Drugs, 19:513-20,2010; Wu etc., Hepatobiliary Pancreat Dis Int, 8:53-58,2009).HUTI is also at other multiple lung, liver, and heart, plays a role in nephropathy infection model (referring to Bao etc., Eur J Pharmacol 603:114-9,2009; Kim etc., Cardiology 114:264-70,2009; Matsuo etc., Thromb Res 52:237-45,1988; Saitoh etc., Anesth Analg 89:1565-9,1999; Mastumoto etc., Masui 38:531-9,1989; Aoike etc., Nephron 52:368-9,1989).In a clinical study, hUTI associating thymosin alpha 1 can increase severe sepsis patient's survival rate (referring to Li etc., JIntensive Care Med, 24:47-53,2009).In another clinical study, the acute lung injury (ALI) that hUTI is caused by SARS virus treatment or acute respiratory distress disease (ARDS) show effectively (referring to US Patent No. 7,470,666).
As far back as 1985, the hUTI of Japanese Mochida company extraction from people's urine just obtained listing license, trade(brand)name " Miraclid ".Its principal indication is the acute circulatory failure due to pancreatitis and shock.Other similar product comprises " injection UTI ", " Ulistin ", " Ustatin " that Korea S Kolon drugmaker produces that Korea S HanLim drugmaker produces that the general biochemical pharmaceuticals in Chinese sky, Guangzhou (Techpool) produces, and " Statin " of the production of Korea S Yu Young drugmaker.At present, hUTI is widely used in treating adjuvant drug in acute attack, acute circulatory failure, disseminated inravascular coagulation (DIC), tumour, shock and the surgical operation of acute pancreatitis, chronic pancreatitis clinically, while preventing and treating cisplatin chemotherapy to the prevention of the assisting therapy of the loss of renal function, AIDS and threatened abortion and treatment etc.
At present, on market, commercial hUTI formulation products all extracts from people's urine, but as the class medicine extracting from people's urine, how to avoid virus to pollute and guarantee that the quality stability of product is very large technical barrier, because the limitation of detection method and inactivation of virus technology and some unpredictable new Causative virus infect, happen occasionally, can not stop to urinate source property product completely the possibility that virus is polluted occurs.In addition also there is the problem that raw material sources are limited, gather difficulty and purge process complexity in urine source hUTI.At present, although urine source hUTI has obtained breakthrough in indication, and gets permission listing in China, Japan, Korea S, in developed countries such as US and Europeans, still do not allow the composition that animal is extracted to carry out clinical trial and final listing as medicine.Comparatively speaking, restructuring hUTI in its purity, antigenicity, security, have advantages of that without aspects such as self urine borne virus infection urine source hUTI is incomparable, shows wide medical prospect.Therefore, by gene recombination technology, obtaining a large amount of high reactivity hUTI albumen is good selection, and not yet has in the world so far restructuring hUTI marketing drugs.
Urinary trypsin inhibitor is used as human cytokines medicine, and indispensable condition is to have correct three-dimensional conformation, has physiologically active and no antigen to reply.For the albumen preparing by gene recombination technology, host should comprise that glycosylation and disulfide linkage form in process of production, and other posttranslational modification processes.The medicinal rhUTI of restructuring preparation need consider the problems referred to above.Along with the widespread use of gene recombination technology, adopt yeast expression system, the rhUTI of purifying has the tryptic activity of inhibition in vitro.Bayer A.G is (referring to US Patent No. 5,407,915) with cereuisiae fermentum, express rhUTI albumen and there is external activity, but its N-end there is serious unhomogeneity problem: 60%hUTI has natural N-terminal, and the few L-Ala (Ala) of 40%N-end.In addition, the restructuring rhUTI albumen of yeast expression, its expression amount is very low, at present the production peak of report be only 55mg/L (referring to Jian-qiu Wang etc., Protein Expr Purif, 60:127-31,2008; US Patent No. 5,407,915).In addition, the rhUTI albumen of yeast expression easily produces the posttranslational modification process that is different from native protein, may cause reduction and/or other side effect of external activity, for example, bring out immune response.Afterwards, there is much research all to show, the rhUTI of bacterial expression has equally and suppresses tryptic effect (referring to Brinkmann T etc., J Biol Chem, 272:11171-11175,1997), but prokaryotic system is not sheared and machining functions albumen, the albumen of its expression has larger difference at the aspects such as structure and activity and native protein, also has the problem that output is too low simultaneously.Therefore, yeast or intestinal bacteria are not the desirable expression systems (referring to Brooks SA, Mol Biotechnol, 28:241-55,2004) of restructuring rhUTI.In fact, industrial only have a minority human cytokines medicine, as albumin (
by Novozymes company, produced) and quick acting insulin analog (
youEli Lilly company produces) because posttranslational modification process is simple, select yeast as expression system.In prior art, though there are some to adopt Yeast system to express the research report of rhUTI, still never the data of rhUTI large-scale production and animal model test aspect is disclosed.
Have that to improve posttranslational modification function be the main cause that mammalian cell is selected as most of biological medicament expressive hosts.On the whole, the recombinant protein by mammalian cell expression has correct three dimensional fold conformation and is similar to the glycosylation modified of native protein molecule.Wherein, Chinese hamster ovary cell (Chinese Hamster Ovary Cell, CHO) be for the most successful host cell of eukaryote exogenous gene expression, existing increasing pharmaceutical protein has obtained high efficient expression therein, a lot of medicines are put on market, as EPO, G-CSF etc.Compare with other expression system, it has many advantages: (1) posttranslational modification function accurately, and the glycosylation pharmaceutical protein of expression is approaching native protein molecule most aspect molecular structure, physicochemical property and biological function; (2) expression product energy exocytosis, is convenient to separation and purification; (3) there is efficient amplification and the ability to express of foreign gene; (4) there are higher tolerance shearing force and osmotic pressure ability, can carry out suspension culture or grow at serum free medium middle-high density, more than volume of culture can reach 1000L; (5) Chinese hamster ovary celI belongs to inoblast, seldom secretes intrinsic protein, is conducive to the separation and purification of foreign protein.Yet, there is the rhUTI reporting by Chinese hamster ovary cell expression can produce a large amount of aggegations, cause active most of forfeiture.Its Protein agglutination is by (referring to Seefeldt MB etc., Protein Sci, 13:2639-50,2004) due to the covalency cross connection between non-natural disulfide linkage between albumen.
In health volunteer, after intravenous injection (i.v.) hUTI 0 to 3 hour, be about 33 minutes its plasma half-life, accounts for 90% injection hUTI and be eliminated.After 4 hours, this stage clearance rate slows down, its transformation period is about 2 hours.After injection 7 hours, residual hUTI only surplus 1% (referring to Jonsson-Berling etc., Scand J Clin Lab Invest, 51:549-57,1991) in blood plasma.The rat of infusion hUTI is carried out to pharmacokinetics and histology and distribute and studies show that, kidney is the main degraded of hUTI and Excretory organ.In addition, adopt the method for immunohistochemical methods, observe hUTI and in Normal human tissue and pathological tissues, distribute and be positioned renal glomerulus (referring to Yoshida etc., Cancer, 64:860-9,1989).Although the purge mechanism of hUTI in blood plasma is still not clear, kidney should be its main katabolism organ.
Because the body-internal-circulation transformation period of hUTI is shorter, and in use security of hUTI is good, and few side effects, in many application documents of current domestic report, only has several examples to occur fash, does not occur anaphylactic shock and important organ infringement.The clinical using dosage of recommendation of commercially available hUTI is 5～20Wan unit, every day 1～3 time.In clinical application, confirm the increasing along with hUTI dosage, its more remarkable treatment effect, and non-evident effect.This makes to develop its long-acting analogue, to reduce administration number of times, reduces medicine fluctuation in vivo, improves patient's compliance.In order to extend the body-internal-circulation transformation period of hUTI, can, by increasing the molecular weight of hUTI, slow down the scavenging(action) of kidney.This strategy is applied to the exploitation of multiple prolonged action preparation, for example increase EPO (
by Amgen company, researched and developed) N-glycosylation site, or by PEG molecule be connected in G-CSF (
by Amgen company, researched and developed).
The immunoglobulin (Ig) of IgG class is rich in protein in human blood.Their transformation period can be up to 21 days, and Fc fragment be in IgG holder compared with long half-lift major cause, there is the effect of stabilize proteins simultaneously.
Restructuring hUTI in its purity, antigenicity, security, have advantages of that without aspects such as self urine borne virus infection urine source hUTI is incomparable, shows good medical prospect.But still have at present that rhUTI expression amount is low, Half-life in vivo is short and the technical problem such as poor activity.
Summary of the invention
The present invention aims to provide a kind of restructuring dimerization hUTI albumen, its preparation method and application, and to solve, in prior art, rhUTI expression amount is low, Half-life in vivo is short and the technical problem of poor activity.
To achieve these goals, according to an aspect of the present invention, provide a kind of restructuring dimerization hUTI albumen.This restructuring dimerization hUTI albumen holds C end to comprise successively the amino acid residue sequence of people UTI, peptide linker and human IgG Fc variant from N, wherein, human IgG Fc variant is selected from following group: human IgG2's hinge region, CH2 and the CH3 region of (i) containing Pro331Ser sudden change; (ii) human IgG 4 hinge regions, CH2 and the CH3 region of containing Ser228Pro and heu235Ala sudden change; (iii) human IgG1's hinge region, CH2 and the CH3 region of containing Leu234Val, Leu235Ala and Pro331Ser sudden change.
Further, peptide linker contains 2-20 amino-acid residue, and peptide linker contains the amino-acid residue that two or more are selected from glycine, Serine, L-Ala and Threonine.
Further, the amino acid residue sequence of peptide linker is as shown in SEQ ID NO:7.
Further, the amino acid residue sequence of restructuring dimerization hUTI albumen is as shown in SEQ ID NO:2,4 or 6.
Further, the amino acid residue sequence of restructuring dimerization hUTI albumen is the aminoacid sequence shown in the SEQ ID NO:2,4 or 6 having removed after the hUTI leading peptide of 1 to 19 amino acids residue.
A kind of DNA sequence dna of the above-mentioned restructuring dimerization hUTI albumen of encoding is provided according to a further aspect in the invention.
Further, this DNA sequence dna has the DNA sequence dna shown in SEQ ID NO:1,3 or 5.
In accordance with a further aspect of the present invention, provide a kind of carrier, this carrier comprises above-mentioned DNA sequence dna.
In accordance with a further aspect of the present invention, provide a kind of host cell, this host cell comprises above-mentioned carrier.
Further, host cell is the derived cell strain of CHO, comprises the Chinese hamster ovary celI strain of above-mentioned carrier.
In accordance with a further aspect of the present invention, provide a kind of pharmaceutical composition, this pharmaceutical composition comprises pharmaceutically acceptable carrier, vehicle or thinner, and the above-mentioned restructuring dimerization hUTI albumen of significant quantity.
In accordance with a further aspect of the present invention, provide a kind of preparation method of above-mentioned restructuring dimerization hUTI albumen, the method is to adopt above-mentioned host cell preparation.
According to another aspect of the invention, above-mentioned restructuring dimerization hUTI albumen is combined the medicine of disease, system inflammation reaction syndromes, shock at the acute attack for the preparation for the treatment of tumour, acute pancreatitis, chronic pancreatitis, acute circulatory failure, disseminated inravascular coagulation, multiple organ dysfunction sexual dysfunction, adjuvant drug in surgical operation, improve blood microcirculation, amelioration of inflammation, while preventing and treating cisplatin chemotherapy to the application in the medicine of the prevention of the assisting therapy of the loss of renal function, AIDS and threatened abortion and treatment.
Below specifically introduce content of the present invention:
Fc element
Fc element is from the Fc region of immunoglobulin (Ig), and Fc has vital role in the immune defense of eliminating pathogen.The effector function of IgG is mediated by Fc, by two kinds of main mechanisms: the combination of (1) and cell surface Fc acceptor (Fc γ Rs), by antibody dependent cellular cytotoxicity (ADCC) approach digestion pathogenic agent, or the combination of the C1q of (2) and the first complement component C1 part, cause cytotoxicity (CDC) approach that depends on complement, thus cracking pathogenic agent.In four kinds of human IgG isotypes, IgG1 and IgG3 can be effectively in conjunction with Fc γ Rs.IgG4 is with the binding affinity of Fc γ Rs than a low order of magnitude of IgG1 and IgG3, and the combination of IgG2 and Fc γ Rs is so low that to be difficult to measure.Human IgG1 and IgG3 can also be effectively in conjunction with C1q, and activating complement cascade reaction.Human IgG2 is fixing very weak to complement, and the extreme ability that lacks activating complement cascade of IgG4 performance (referring to Jefferis R etc., Immunol Rev, 163:59-76,1998).For medical use, the Fc region of restructuring dimerization albumen must not can mediate effector function and cracking or remove these cells.Therefore, the Fc region of hUTI-L-Fc must be non-cracking performance, and in conjunction with Fc γ Rs and C1q and aspect trigger effect subfunction, Fc is non-activity preferably.Obviously, do not have a kind of natural IgG isotype to be applicable to producing hUTI-L-Fc restructuring dimerization albumen.In order to obtain the Fc of non-cracking performance, must make some amino acid mutations in natural Fc region, to reduce its effector function.
By comparing the aminoacid sequence of the IgG isotype of people and mouse, near the Fc partial display N-terminal of CH2 region works in the combination of IgG Fc and Fc γ Rs.Genetic engineering antibody is verified at 234 importance Duncan AR to 237 motifs etc., Nature, and 1988,332:563-564).Press the people such as Kabat (referring to Sequences of Proteins of Immunological Interest, the 5th edition, United States Department of Health and Human Services, 1991) described EU number system is numbered amino-acid residue.In four kinds of human IgG isotypes, the combination of IgG1 and IgG3 and Fc γ Rs is best, and has identical sequence Leu234-Leu-Gly-Gly237 (Fig. 1 has only shown IgG1).With low-affinity with IgG4 that Fc γ Rs is combined, its sequence contains single amino acids and replaces, Phe replaces the Leu of 234.In the IgG2 in conjunction with Fc γ Rs not, two replacements and a disappearance form Val234-Ala-Gly237 (Fig. 1).Active in order to reduce the combination of Fc and Fc γ Rs and ADCC, with Ala, substitute Leu235 in IgG4 (referring to Hutchins JT etc., Proc Natl Acad Sci USA, 92:11980-11984,1995).Glu233-Leu-Leu235 sequence in IgG1 antibody was once replaced by the Pro233-Val-Ala235 correlated series with in IgG2.This change has lost IgG1 variant to see through Fc γ R-mediation to remove the ability of target cell (referring to Isaacs JD etc., J Immunol, 161:3862-386,1998) in mouse.
For Fc γ R, be combined very important second section with C1q and be positioned at (Duncan AR etc., Nature, 332:738-740,1988) near the CH2 region carboxyl terminal of human IgG.In four kinds of human IgG isotypes, in this part, only there is a site to show and replace: with the Ala330 in IgG1, IgG2 and IgG3 and Pro331, substitute Ser330 and the Ser331 (Fig. 1) in IgG4.The existence of Ser330 does not affect the combination of Fc γ R and C1q.With Ser, substitute Pro331 and make IgG1 lose the binding affinity with C1q, and with Pro substitute Ser331 partly retained IgG4 complement fixation active (referring to Tao MH etc., J ExpMed, 178:661-667,1993; Xu Y etc., J Biol Chem, 269:3469-3474,1994).
Peptide linker
The length of connection peptides is extremely important to the activity of restructuring dimerization albumen.Existing people has reported erythropoietin (EPO) derivative (as dipolymer), compare with EPO monomer, the restructuring dimerization albumen that contains 2 complete EPO regions (3 to 7 the amino acid peptide joints of being separated by) shows the activity that weakens (referring to Qiu H etc., J Biol Chem, 273:11173-11176,1998).Yet when the length of the interregional peptide linker of these two EPO is 17 amino acid, the in vitro and in vivo biological activity of dimer EPO molecule obviously improves (Sytkowski AJ etc., J Biol Chem, 274:24773-24778,1999; U.S. Patent No. 6,187,564).The connection peptides of this possible explanation for increasing between restructuring dimerization albumen two portions, makes two portions of this molecule can exercise respectively its function (referring to Ashkenazi A etc., Curr Opin in Immunol, 9:195-200,1997).
The inventor is through long-term and deep research, and the hinge region peptide linker that has designed first a kind of uniqueness reduces space steric effect, can make the C end of hUTI and the restructuring dimerization albumen of Fc variant coupling, and there is soft peptide linker centre.This restructuring dimerization albumen not only can not cause the afunction of hUTI, can maintain on the contrary, even improve the biological activity of hUTI-Fc restructuring dimerization albumen.The amino acid residue sequence of preferred peptide joint is GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.
In addition, the present invention also finds, the peptide linker adding between hUTI and human IgG Fc variant improves the Bioactivity of hUTI-L-vFc in two ways: (1) makes Fc region away from the structural domain on hUTI, (2) make a hUTI away from the structural domain of another hUTI, thereby reduce space steric effect.And people's IgG Fc variant contains amino acid mutation in CH2 region in 228,234,235,331 sites, thus the effector function of reduction Fc.
Restructuring dimerization albumen and preparation method thereof
The present invention's dimerization albumen of recombinating is produced by Chinese hamster ovary celI, through stdn purifying procedure, prepares.According to nucleotide sequence of the present invention, the art personnel can make coding nucleic acid of the present invention with various currently known methodss easily.These methods are such as but not limited to PCR, DNA synthetic etc., and concrete method can be referring to J. Pehanorm Brooker, < < molecular cloning experiment guide > >.As one embodiment of the present invention, the method for carrying out again the splicing of gene subclone by salvage nucleotide sequence builds full-length gene nucleic acid sequence encoding of the present invention.
The present invention also provides a kind of expression vector, comprises the encode DNA sequence dna of restructuring dimerization albumen of the present invention and the connected expression regulation sequence of operability with it.Described " operability is connected " or " being operationally connected in " refer to a kind of like this situation, and some part of linear DNA sequence can regulate or control the activity of same linear DNA sequence other parts.For example, if the transcribing of promotor control sequence, it is exactly to be operationally connected in encoding sequence so.
Expression vector can adopt commercially available such as but not limited to: pcDNA3, pIRES, pDR, pBK, pSPORT etc. can be used for the carrier that eukaryotic cell system is expressed.Those skilled in the art can select suitable expression vector according to host cell.
According to the restriction enzyme mapping of known unloaded expression vector, those skilled in the art can shear and splicing by Restriction Enzyme according to ordinary method, the DNA encoding sequence of restructuring dimerization albumen of the present invention is inserted to suitable restriction site, obtain recombinant expression vector of the present invention.
The present invention also provides and has expressed the recombinate host cell of dimerization albumen of the present invention, wherein contains the DNA encoding sequence of restructuring dimerization albumen of the present invention.Described host cell is eukaryotic cell preferably, such as but not limited to CHO, and COS cell, 293 cells, RSF cell etc.As optimal way of the present invention, described cell is Chinese hamster ovary celI, and it can preferably express restructuring dimerization albumen of the present invention, can obtain physiologically active good, the restructuring dimerization albumen having good stability.
The present invention also provides a kind of and prepares the recombinate method of dimerization albumen of the present invention by recombinant DNA technology, and its step comprises:
1) provide the nucleotide sequence of coding restructuring dimerization albumen;
2) by 1) nucleotide sequence be inserted into suitable expression vector, obtain recombinant expression vector;
3) by 2) recombinant expression vector import suitable host cell;
4) cultivate under conditions suitable for the expression host cell;
5) stable cell lines of screening high expression level;
6) collect supernatant liquor, and purification of Recombinant dimerization protein product.
Described encoding sequence is imported to the multiple known technology that host cell can adopt this area, such as but not limited to: calcium phosphate precipitation, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription method, phage transduction method, alkalimetal ion method.
About cultivation and the expression of host cell can be referring to Olander RM Dev Biol Stand 1996; 86:338.Can, by cell and residue in centrifugal removal suspension, collect supernatant liquor.Can identify by agarose gel electrophoresis technology.
The character that can be basic homogeneous by the above-mentioned restructuring dimerization protein purification preparing, for example, be single band on SDS-PAGE electrophoresis.For example, when recombinant protein is secreting, expressing, can adopt commercial ultra-filtration membrane to carry out separated described albumen, the product such as the company such as Millipore, Pellicon, first will express supernatant and concentrate.The method that concentrated solution can adopt gel chromatography is purifying in addition further, or adopts the method purifying of ion exchange chromatography.Such as anion-exchange chromatography (DEAE etc.) or cation-exchange chromatography.Gel matrix can be the matrix that agarose, dextran, polymeric amide etc. are usually used in protein purification.Q-or SP-group are comparatively desirable ion-exchange groups.Finally, available hydroxyapatite adsorption chromatography also, metal chelate chromatography, the methods such as hydrophobic interaction chromatography and RPLC (RP-HPLC) are to the further refining purifying of above-mentioned purified product.Above-mentioned all purification steps can utilize different combinations, finally make purity of protein reach basic homogeneous.
Can utilize the affinity column of the specific antibody, acceptor or the part that contain described restructuring dimerization albumen to carry out purifying to the restructuring dimerization albumen of expressing.According to the characteristic of used affinity column, can utilize conventional method, as the amalgamation polypeptide of method elution of bound on affinity column such as high-salt buffer, change pH.Selectively, aminoterminal or the carboxyl terminal of described restructuring dimerization albumen also can contain one or more polypeptide fragments, as albumen label.Any suitable label may be used to the present invention.For example, described label can be FLAG, HA, c-Myc, 6-His or 8-His etc.These labels can be used for restructuring dimerization albumen to carry out purifying.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical composition, and it contains significant quantity (as 0.000001-90wt%; 0.1-50wt% preferably; Better, restructuring dimerization albumen of the present invention 5-40wt%), and pharmaceutically acceptable carrier.Conventionally, that the dimerization albumen of the present invention of significant quantity can being recombinated is formulated in is nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, preferably, pH is about 6-8.Term " significant quantity " or " effective dose " refer to and can produce function or amount active and that can be accepted by people and/or animal to people and/or animal.The composition of " pharmaceutically acceptable " is applicable to people and/or Mammals and without excessive bad side reaction (as toxicity, stimulation and transformation reactions), has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle and thinner.
Pharmaceutically acceptable carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Conventionally pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into injection form, for example, with physiological saline or contain glucose and the aqueous solution of other assistant agents is prepared by ordinary method.Described pharmaceutical composition should be manufactured under aseptic condition.The dosage of activeconstituents is treatment significant quantity.Pharmaceutical preparation of the present invention also can be made into sustained release preparation.
The significant quantity of restructuring dimerization albumen of the present invention can change with severity of the pattern of administration and disease to be treated etc.The selection of preferred significant quantity can be determined (for example, by clinical trial according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization ratio of described restructuring dimerization albumen, metabolism, transformation period etc.; The severity of the disease that patient will treat, patient's body weight, patient's immune state, the approach of administration etc.
A kind of typical embodiment according to the present invention, restructuring dimerization hUTI albumen holds C end to contain successively people UTI, peptide linker and human IgG Fc variant (being expressed as hUTI-L-vFc) from N.Wherein, IgG Fc variant is without cracking performance, and compares and contain amino acid mutation with natural IgG Fc.Preferably, wherein people Ig Fc variant contains and is selected from following variant hinge region, CH2 and CH3 region: the human IgG l that (A) contains Leu234Val, Leu235Ala and Pro331Ser sudden change; (B) human IgG2 of containing Pro331Ser sudden change; (C) human IgG 4 that contains Ser228Pro and Leu235Ala sudden change.Preferably, use an about 2-20 amino acid length, contain the flexible peptide linker that following 2 kinds or multiple amino acids form: glycine, Serine, L-Ala and Threonine, if disclosed preferred sequence in one embodiment of the invention is SEQ ID:7 (GlySerGlyGlyGlySerGlyGlyGly GlySerGlyGlyGlyGlySer).
Preferably, the aminoacid sequence of restructuring dimerization hUTI albumen of the present invention is as shown in SEQ ID NO:2,4 or 6, its maturation protein is for having removed hUTI leading peptide (1 to the 19 amino acids residue) aminoacid sequence shown in SEQ ID NO:2,4 or 6 afterwards, wherein, people Ig Fc variant contains hinge region, CH2 and CH3 region.Amino acid mutation is contained 228,234,235 and 331 (positions of being determined by EU number system) in its CH2 region, thereby reduces the effector function of Fc.
A kind of typical embodiment according to the present invention, provides a kind of DNA sequence dna of the above-mentioned restructuring dimerization hUTI albumen of encoding.Preferably, this DNA sequence dna has the DNA sequence dna shown in SEQ ID NO:1,3 or 5.
According to the present invention, a kind of typical embodiment, provides a kind of carrier, and this carrier comprises above-mentioned DNA sequence dna.
According to the present invention, a kind of typical embodiment, provides a kind of host cell, and this host cell comprises above-mentioned carrier.Preferably, host cell is the derived cell strain of CHO.
A kind of typical embodiment according to the present invention, pharmaceutical composition of the present invention comprises pharmaceutically acceptable carrier or vehicle or thinner, and the restructuring dimerization hUTI albumen of the present invention of significant quantity.
Restructuring dimerization hUTI albumen of the present invention is usually applied to adjuvant drug in acute attack, acute circulatory failure, tumour, shock and the surgical operation of acute pancreatitis, chronic pancreatitis, while preventing and treating cisplatin chemotherapy to the prevention of the assisting therapy of the loss of renal function, AIDS and threatened abortion and treatment etc.
A kind of typical embodiment according to the present invention, provides a kind of from mammal cell line clone preparation as derivative in CHO or the method for producing this restructuring dimerization hUTI albumen.Cultivate the clone of transfection, the dimerization hUTI albumen that makes to recombinate be take over 100 (best is 300) μ g/10 in its growth medium during 24 hours
6under the level of (1,000,000) individual cell, express.These hUTI-L-vFc dimer proteins demonstrate serum half-life in the in vitro and in vivo biological activity of height and the body of prolongation and without adverse side effect, thereby improved pharmacokinetics and drug effect, and then reduced and realized the required dosage of similar drug effect and frequency injection.
In addition, the preparation method of restructuring dimerization hUTI albumen disclosed in this invention, expression output is high and because of the coupling with IgG Fc variant, formed restructuring dimerization albumen can obtain efficiently purifying easily by Protein A affinity chromatography.In one embodiment of the present invention, Chinese hamster ovary celI strain is cultivated 15 days in 100mL shaking flask, the restructuring dimerization albumen cumulative withdrawal of its expression is 3g/L (Fig. 6).Between the 5th day to the 11st day of cell cultures, viable cell number is about at most 6 * 10
6individual/mL, with this understanding, secretion rate is determined as 50 μ g/10
6individual cell/24 hour.
The advantage of restructuring dimerization hUTI albumen of the present invention and preparation method thereof is summarized as follows:
The dimer protein that 1.Fc and hUTI coupling form has very high expression amount in Chinese hamster ovary celI, higher 30 times than hUTI expression amount in Chinese hamster ovary celI, higher 60 times than the expression amount in yeast cell.
Purifying process efficiently convenient, greatly reduce production costs.
3. expressed restructuring dimerization hUTI albumen and the natural hUTI of the present invention has similar In vitro biological activity and exceeds the Biological acdtivity in vivo (mol ratio is active) of 38 times.
4. the expressed restructuring dimerization hUTI proteoplast internal recycle Increased Plasma Half-life of the present invention, the minimizing of serum Chinese traditional medicine fluctuation of concentration, security improves, and the improvement of tolerance reduces frequency of injection and improves patient's quality of life.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and can combining mutually between specifically described each technical characterictic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, at this, tire out and state no longer one by one.
Accompanying drawing explanation
Fig. 1 has shown the comparison of the hinge region of human IgG1, IgG2, IgG4 and their variants and the aminoacid sequence in CH2 region.Compare this three partial amino-acid series: amino acid region 228,234-237 and 330-331.The amino acid mutation of these variants shows with bold Italic.Amino-acid residue numbering is to demarcate according to EU number system.
Fig. 2 has shown the nucleotide sequence (with SEQ ID:1) of the hUTI-L-vFc γ 2 of HindIII-EcoRI fragment in PCDNA3 expression vector and the aminoacid sequence (with SEQ ID:2) of deriving.HUTI consists of signal peptide (1-19), ripe hUTI albumen (20-162).Ripe restructuring dimerization albumen contains hUTI (20-162), peptide linker (163-178) and Fc γ 2 variants (179-401).
Fig. 3 has shown the nucleotide sequence (with SEQ ID:3) of the hUTI-L-vFc γ 4 of HindIII-EcoRI fragment in PCDNA3 expression vector and the aminoacid sequence (with SEQ ID:4) of deriving.HUTI consists of signal peptide (1-19), ripe hUTI albumen (20-162).Ripe restructuring dimerization albumen contains hUTI (20-162), peptide linker (163-178) and Fc γ 4 variants (179-407).
Fig. 4 has shown the nucleotide sequence (with SEQ ID:5) of the hUTI-L-vFc γ 1 of HindIII-EcoRI fragment in PCDNA3 expression vector and the aminoacid sequence (with SEQ ID:6) of deriving.HUTI consists of signal peptide (1-19), ripe hUTI albumen (20-162).Ripe restructuring dimerization albumen contains hUTI (20-162), peptide linker (163-178) and Fc γ 1 variant (179-405).
Fig. 5 has shown the gene mapping of the eukaryon expression plasmid of constructed hUTI-L-vFc restructuring dimerization protein gene.This expression plasmid total length 9063bp, contains 10 oligogene segments, comprises 1.hCMV promotor; 2. target gene hUTI-L-vFc; 3.EMCV IRES; 4.mDHFR screening-gene; 5.bGH pause sequence; 6.SV40 promotor; 7. kalamycin resistant gene; 8.SV40 pause sequence; 9.ColE1 replicon; 10. ampicillin resistance gene.
Fig. 6 has shown the growth of 300ml shaking flask inner cell strain and the concentration trend curve figure of secretion hUTI-L-vFc albumen thereof.
Fig. 7 shown restructuring hUTI-L-vFc purifying protein (by
represent) with natural urine source hUTI purifying protein (by ● represent) external activity comparison.The IC50 value of UTI is 81.5 ± 6.0nM, and the IC50 value of UTI-Fc is 70.9 ± 1.5nM.
Fig. 8 has shown the activity in vivo comparison of the acute pancreatitis mice animal model that restructuring hUTI-L-vFc purifying protein and natural urine source hUTI induce at tree toad element.Urine source hUTI just can see the serum amylase faint restraining effect (8%) of rising stage sharply at high dosage (40mg/Kg) very, cannot see any restraining effect when low dosage (1mg/Kg); And hUTI-L-vFc just sees the serum amylase remarkable restraining effect of rising stage (26% sharply at low dosage (3mg/Kg), p < 0.005), restraining effect more obviously (53%, p < 0.001) when median dose (30mg/Kg).Consider the difference of their molecular sizes, the Dosages of recombinant dipolymer hUTI-L-vFc than urine source hUTI Dosages low 38 times, and drug effect is better.
Fig. 9 has shown the 10%SDS-PAGE protein electrophoresis collection of illustrative plates of hUTI-L-vFc purifying protein (Lane 2) with natural urine source hUTI (lane 3).
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1. builds the expression vector of coding hUTI-L-vFc restructuring dimerization albumen
The gene of coding hUTI leading peptide and maturation protein adopts the way of synthetic to obtain, the 506bp DNA fragmentation of synthesized is inserted to transfer vector as between the EcoRV restriction enzyme site in pUC57, obtained phUTI plasmid, the sequence of its contained hUTI gene can be verified by DNA sequencing.
Flexible peptide Linker and human IgG Fc region Fc
γ 2variant vFc
γ 2(Pro331Ser sudden change), Fc
γ 4variant vFc
γ 4(Ser228Pro and Leu235Ala sudden change), Fc
γ 1variant vFc
γ 1the fusion gene of (Leu234Val, Leu235Ala and Pro331SSer sudden change) is obtained by the method for synthetic, and the fragment 5 ' of synthesized and 3 ' end respectively have a Restriction Enzyme inscribe site, are respectively BamHI and EcoRI.The DNA fragmentation of acquisition is inserted into transfer vector as between the BamHI of PUC19 and EcoRI site, obtains PL-vFC γ plasmid.By DNA sequencing checking L-vFc
γgene order.For preparation hUTI-L-vFc γ fusion gene, phUTI plasmid is carried out to double digestion with SpeI and BamHI, after electrophoresis, glue reclaims hUTI fragment again.Purified hUTI fragment is inserted into 5 '-end of peptide linker in PL-vFC γ plasmid, connects and composes phUTI-L-vFc γ plasmid.The peptide linker that constructed fusion gene is comprised of hUTI, Gly-Ser and Fc γ variant gene form.
The gene order of the complete coding hUTI-L-vFc γ restructuring dimerization albumen obtaining, after HindIII/EcoRI double digestion, is inserted into mammalian cell expression plasmid as between the corresponding restriction enzyme site of PCDNA3 (Invitrogen).As shown in Figure 5, the final fusion gene expression plasmid pCDNA3-hUTI-L-vFc γ obtaining, this plasmid is containing cytomegalovirus early promoter, and it is the required enhanser of mammalian cell high level expression foreign gene.This plasmid also contains selected marker thing, thereby can have amicillin resistance in bacterium, and can have G418 resistance in mammalian cell.In addition, when host cell is DHFR genetic expression defective type, the Tetrahydrofolate dehydrogenase of the mouse that PCDNA3 expression vector is contained (DHFR) gene, make it when methotrexate (MTX) exists, energy coamplification hUTI-L-vFc γ fusion gene and DHFR gene (United States Patent (USP) 4,399,216).
The peptide linker of (and each other Chemical bond) existing between people UTI and Fc part (being preferably flexible joint) has increased the flexible of UTI region and has improved its biological activity.For the purpose of the present invention, preferably length is approximately 20 or (but can not be less than 2) amino acid whose peptide linker still less, certainly by the peptide linker of 1 Amino acid profile also in protection scope of the present invention.Should use contain or by 2 or more multiselect from the peptide linker of following Amino acid profile: glycine, Serine, L-Ala and Threonine.The example of peptide linker contains a Gly-Ser peptide member, as GlyGlyGlyGlySer.Fig. 2, Fig. 3, Fig. 4 have shown respectively containing three kinds of Fc
γthe nucleotide sequence of variant fusion gene and the aminoacid sequence of derivation, they jointly contain encoding human UTI, 16-amino acid peptide joint (GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer) and contain separately coding Fc
γ 2variant vFc
γ 2(hUTI-L-vFc γ 2), Fc
γ 4variant vFc
γ 4(hUTI-L-vFc γ
4) or Fc
γ 1variant vFc
γ 1(hUTI-L-vFc γ
1) sequence.
The hUTI-L-vFc adopting during specific experiment in embodiment 2-6 is hUTI-L-vFc
γ2, certainly can use the same method claimed other restructuring dimerizations hUTI of the present invention is operated.
Embodiment 2. expression of restructuring dimerization albumen in stably transfected cell line
By the expression vector plasmid pCDNA3-hUTI-L-vFc of restructuring
γ2 are transfected into mammalian host cell line, to express hUTI-L-vFc
γ2 restructuring dimerization albumen.In order to stablize high-caliber expression, preferred host cell is to be DHFR deficient CHO-cell (U.S. Patent No. 4,818,679).Preferred transfection method is an electroporation, also can use other method, comprises calcium phosphate cosedimentation, fat transfection and Protoplast fusion.In electroporation, with being set to the Gene Pulser Flectroporator (Bio-Rad Laboratories, Hercules, CA) of 250V electric field and 960 μ Fd electric capacity, in cuvette 2～5 * 10
7in individual cell, add the linearizing plasmid DNA of PvuI for 10 μ g.In transfection two days later, substratum is made into the growth medium containing 0.6mg/mL G418.By the elisa assay method of anti-human IgG Fc, screening is to selecting medication to have the transfectant of resistance.Also the elisa assay method of available anti-hUTI is expressed the quantitative of restructuring dimerization albumen.By Method of Limited Dilution 96Kong tissue culturing plate, subclone produces the hole of high-level Fc restructuring dimerization albumen.
The production of embodiment 3. restructuring hUTI-L-vFc dimerization albumen
In order to realize the high-caliber expression of restructuring dimerization albumen, should carry out coamplification with the DHFR gene that suppressed by MTX medicine.In the growth medium that contains progressive concentration MTX, with the restructuring dimerization protein gene of DHFR gene coamplification transfection.The transfectant that can grow in up to 1 μ g/mL MTX substratum with Method of Limited Dilution method subclone.The secretion rate of subclonal cell line is done further to analyze.Screening secretion level surpasses approximately 100 (preferably approximately 200) μ g/10
6the clone of (1,000,000) individual cell/24 hour, makes its suspension growth in serum free medium.
The high yield cell strain that the present invention screens first carries out serum-free domestication and cultivates in culture dish, then transfers in shaking flask and suspends and tame cultivation.In 100mL shaking flask, cultivate 15 days, Fig. 6 has shown that the restructuring dimerization albumen cumulative withdrawal of its expression is 3g/L.Between the 5th day to the 11st day of cell cultures, viable cell number is about at most 6 * 10
6individual/mL, with this understanding, secretion rate is determined as 50 μ g/10
6individual cell/24 hour.In order to obtain more hAT-L-vFc recombinant protein, also can select 2000ml shake-flask culture.Finally use conditioned medium purification of Recombinant dimerization albumen.
Purifying and the qualitative analysis of embodiment 4. restructuring hUTI-L-vFc dimerization albumen
With 1N NaOH, the conditioned medium that contains restructuring dimerization albumen is titrated to pH 7～8, then with the nitrocellulose strainer of 0.45 micron, filters.Filtrate is loaded onto on the ProsepA post of phosphate buffer saline (PBS) balance.Dimerization protein binding to be reorganized, after Prosep A, discards the component of outflow.With PBS, wash this post, until the OD value at 280nm place is lower than 0.01.The restructuring dimerization albumen of the citrate buffer solution elution of bound that is then 3.75 with 0.1M pH.1M K with 0.4 volume
2hPO
4neutralization, merges the component that contains purifying protein, and dialyses with PBS.Then with the nitrocellulose strainer of 0.22 micron, filter, and be stored in-70 ℃.As shown in Figure 9, the urine source hUTI being recorded by SDS-PAGE (ulinastatin, day general biochemistry) molecular weight (Lane3) is shown as 38kD.Under the same conditions, purified restructuring hUTI-L-vFc (Lane2) migrates to about 55kD place (thick band), because of the otherness of glycosylation form, separately has other high-molecular-weight proteins of small amount to migrate to about 70kD place (slice band).With BSA as standard, by quantitative this restructuring dimerization albumen of BCA protein analysis.
Embodiment 5. external activities detect
In the present invention, in Chinese hamster ovary celI culture supernatant, the biologic activity of restructuring hUTI-L-vFc albumen that secrete or purified adopts the recognized standard working method in prior art to measure (referring to Kakade etc., Cereal Chem, 46:518-526,1969), judging by tryptic residual activity in mensuration system.In reaction system with BAPNA (N α-Benzoyl-L-arginine 4-nitroanilide hydrochloride, Sigma-Aldrich company) as pig trypsin Sigma-Aldrich company) substrate.In reaction mixture, comprise the hUTI-L-vFc restructuring dimerization albumen of the diluted hUTI albumen of 20 μ l, purifying or the culture supernatant of hUTI-L-vFc transformant, 5 μ l trypsin 100mg/ml, are dissolved in the CaCl containing 2.94g/L
21mM HCl in), with 0.2M trolamine damping fluid, being adjusted to final volume is 125 μ l.Reaction mixture is hatched to 20min in 37 ℃ of shaking tables, (BAPNA of 25mg is dissolved in the DMSO of minimum volume to add 5 μ l BAPNA, and regulate final concentration to 10mg/ml with 0.2M trolamine damping fluid), then in 37 ℃ of shaking tables, continue to hatch 20min.30% (v/v) Glacial acetic acid termination reaction that adds 30 μ l.Reaction needs to arrange blank and trypsinase contrast simultaneously.At 405nm place, read its light absorption value.Fig. 7 has shown the volumetric molar concentration (nM) of restructuring hUTI-L-vFc dimerization albumen and the corresponding relation of its reference activity of urine source hUTI or purifying.Under these conditions, the IC of hUTI
50value is about 81.5 ± 6.0nM, and purified hUTI-L-vFc albumen is about 70.9 ± 1.5nM.
Embodiment 6. activity in vivo detect
The acute pancreatitis mice animal model of the Caerulein for activity in vivo of the restructuring hUTI-L-vFc albumen of purifying in the present invention (tree toad element) induction is assessed (referring to Ding etc., Hepatobiliary Pancreat.Dis.Int., 9:645-650,2010; Galuppo etc., Br J.Pharmacol., 2011; 162:1186-1201).Tree toad element injection is a kind of relatively more conventional at present method of setting up the animal model of acute pancreatitis.Be characterized in not needing operation, less, easy and simple to handle on organismic internal environment impact, be easy to copy, reproducible; Induced Acute pancreatitis is similar to people's acute pancreatitis on form, biochemical change, time course.Caerulein is the analogue of cholecystokinin, can activate cck receptor.Cck receptor is g protein coupled receptor, after activation, can cause that in pancreatic acinar cell smooth surfaced endoplasmic reticulum, calcium ion discharges by PLC-IP3 signal path, and then activation NADPH sudismase, ROS is generated to be increased, I κ B α phosphorylation, finally causes NF κ B to activate, and inflammation-related factor is as TNF α, IL-6, NO etc. express increase.The interaction of a large amount of inflammation-related factor promotes progressively development system inflammatory reaction of pancreas local inflammation (multiple organ failure syndromes).Therefore the response to oxidative stress that ROS causes is considered to the important pathogenic link of acute pancreatitis development.30 of C57BL/6 male mices getting 8 week age in experiment, 20-22g, is divided into 5 groups, 6/group at random.Fasting in advance be can't help water 15 hours.Each organizes the 1 hour caerulein 50 μ g/kg in interval, injects continuously 6 times.Each group is injected after caerulein with the 5th at the 1st time respectively, the urine source hUTI (ulinastatin) of tail vein injection various dose or restructuring hUTI-L-vFc.The different time points of each treated animal after last injection caerulein got blood approximately 200 μ l from mouse orbit.Blood room temperature is transferred and is set to 0 .5 hour, and centrifugal 10 minutes of 6000rpm, prepares serum.-70 ℃ frozen.Sample is delivered to new drug Research on Safety Assessment center, national Shanghai, detects the content of serum amylase with Hitachi's 7060 full-automatic biochemical detectors, assesses the action effect of tested medicine.In above-mentioned chmice acute induction pancreatitis animal model, serum amylase is injected the last time tree toad element and is risen rapidly afterwards, after 3 hours, reaches peak value, then slowly falls after rise, after 24 hours, gets back to starting point.According to our experimental observation, injecting for the last time tree toad element latter 1 hour is optimum medicine efficacy observation time point.Urine source hUTI just can see the serum amylase faint restraining effect (8%) of rising stage sharply at high dosage (40mg/Kg) very, cannot see any restraining effect when low dosage (1mg/Kg); And hUTI-L-vFc just sees the serum amylase remarkable restraining effect of rising stage (26% sharply at low dosage (3mg/Kg), p < 0.005), restraining effect more obviously (53%, p < 0.001) when median dose (30mg/Kg).The theoretical molecular of recombinant dipolymer hUTI-L-vFc is 5 times of urine source monomer hUTI, if consider that both possible degree of glycosylation are different, from Fig. 9, the actual molecular weight of recombinant dipolymer hUTI-L-vFc is approximately 3 times of urine source hUTI.According to the difference of above molecular weight, the restructuring hUTI-L-vFc that is equal to urine source hUTI mole number very high dosage (120mg/Kg) cannot implement in experiment mice.Consider the huge difference of their molecular sizes, the Dosages of recombinant dipolymer hUTI-L-vFc than urine source hUTI Dosages low 38 times, and drug effect is better.
The pharmacokinetics of embodiment 7. fusion roteins is measured
SD rat single dose (2mg/Kg) tail vein injection hUTI-L-vFc sample, chooses the blood sample that different time points extracts SD rat, anticoagulant heparin, and the supernatant liquor after centrifugal is measured the fusion rotein content in blood plasma by ELISA method.While measuring with ELISA, with the how anti-monoclonal antibodies that carry out the mouse-anti people hUTI of embedding, horseradish peroxidase-labeled of goat-anti people's Fc, detect.In embodiment 4, be about 910 minutes the plasma half-life of purifying hUTI-L-vFc sample, and the purifying urine source hUTI transformation period is about 182 minutes, so the hUTI-L-vFc Half-life in vivo of recombinating in the present invention significantly extends.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (8)
1. a restructuring dimerization hUTI albumen, is characterized in that, it is the amino acid residue sequence of people UTI, peptide linker and human IgG Fc variant successively that described restructuring dimerization hUTI albumen is held C end from N,
Wherein, described human IgG Fc variant is selected from following group:
(i) human IgG2's hinge region, CH2 and the CH3 region of containing Pro331Ser sudden change;
(ii) human IgG 4 hinge regions, CH2 and the CH3 region of containing Ser228Pro and Leu235Ala sudden change;
(iii) human IgG1's hinge region, CH2 and the CH3 region of containing Leu234Val, Leu235Ala and Pro331Ser sudden change;
The amino acid residue sequence of described peptide linker is: GlySerGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer.
2. restructuring dimerization hUTI albumen as claimed in claim 1, is characterized in that, the amino acid residue sequence of described restructuring dimerization hUTI albumen is as shown in SEQ ID NO:2,4 or 6.
3. the DNA sequence dna of the restructuring dimerization hUTI albumen of coding as described in any one in claim 1 to 2.
4. DNA sequence dna as claimed in claim 3, is characterized in that, the DNA sequence dna of described DNA sequence dna as shown in SEQ ID NO:1,3 or 5.
5. a carrier, is characterized in that, comprises the DNA sequence dna as described in claim 3 or 4.
6. a host cell, is characterized in that, comprises carrier as claimed in claim 5.
7. host cell as claimed in claim 6, is characterized in that, described host cell is the derived cell strain of CHO.
8. a pharmaceutical composition, is characterized in that, comprises pharmaceutically acceptable carrier, vehicle or thinner, and the restructuring dimerization hUTI albumen described in any one claim in the claim 1-2 of significant quantity.
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| JP2013102039A JP2013253079A (en) | 2012-05-14 | 2013-05-14 | BIOLOGICALLY ACTIVE Fc FUSION PROTEIN OF HUMAN URINARY TRYPSIN INHIBITOR, AND PREPARATION METHOD AND APPLICATION THEREOF |
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