CN102260342A - Chemical conjugate of I type recombinant deletion human keratinocyte growth factor - Google Patents
Chemical conjugate of I type recombinant deletion human keratinocyte growth factor Download PDFInfo
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Abstract
The invention discloses a conjugate obtained by using a polyethylene glycol molecule to chemically modify an I type recombinant deletion human keratinocyte growth factor. The conjugate is characterized in that an active polyethylene glycol molecule with molecular weight of 5-40 K is chemically coupled with free sulfydryl of a 17th cysteine of the I type recombinant deletion human keratinocyte growth factor (delta 23 KGF-I) and then separated and purified through column chromatography so as to obtain the polyethylene glycol keratinocyte growth factor, wherein 23 amino acids are deleted at a N end of the having 23 amino acids deleted at a N end. By using the conjugate, the metabolism dynamic characteristics in human body are changed while the biological effect of the human keratinocyte growth factor is kept; and prevention and treatment values are obtained for gastrointestinal mucosal injury or ulcerative catarrh caused by chemoradiotherapy and acute chemical liver injury.
Description
Technical field
The present invention relates to the genetically engineered field, be specifically related to the reorganization preparation of deletion human keratinocyte growth factor I type, deletion human keratinocyte growth factor I type polyethyleneglycol modified, and the purifying after modifying and its application.
Background technology
Up to now, found two kinds of keratinocyte growth factors (keratinocyte growth factor, KGF), i.e. keratinocyte growth factor I type (KGF-I) and keratinocyte growth factor II type (KGF-II).They are respectively fibroblast growth factor (fibroblast growth factor, FGF) member of family, again called after FGF-7 and FGF-10 (Aaronoson SA.et al.J.Annals of the New York Academy of science, 1991,638:62-77.).KGF-I is at first by people such as Rubin (Rubin, J.S., et al.Proc Natl Acad Sci U S A, 1989.86 (3): p.802-6.) from the fibroblastic culture supernatant of HFL, separate and purifying, and find to have the effect that promotes the mouse mitosis of horny cell, be keratinocyte growth factor so it is named.KGF-II is Emoto etc. (Emoto H Tagashira S MatteiMG, Journal of biology chemistry, 1997 (37)) isolated coding people KGF-II from lung in 1997 cDNA.
(human keratinocyte growth factor hKGF-I) is positioned at karyomit(e) No. 15 to human keratinocyte Growth Factor I type, and 194 amino-acid residues of its cDNA coding comprise one section 21 amino acid whose secreting signal peptide.Sophisticated hKGF-I one contains the single chain polypeptide of 163 amino-acid residues.Preclinical study and clinical studies show, human keratinocyte Growth Factor I type is a kind of multifunctional growth factor, repair and induce the expression that active oxygen is had an enzyme of function of detoxification to strengthen the epithelium function by propagation, migration, differentiation, survival, the DNA that promotes cell, thereby protective epithelium avoids the damage (Finch of toxicant, P.W., et al., Science, 1989.245 (4919): p.752-5.; Rubin, J.S., et al.V; Farrell, C.L., et al., Int J Radiat Biol, 1999.75 (5): p.609-20.; Farrell, C.L., et al., Cancer Res, 1998.58 (5): p.933-9.; Ellison CA, et al., J Clin Immunol, 2008,28 (5): 600-615.).
Recombinant human keratinocyte growth factor Palifermin (trade(brand)name Kepivance) by the exploitation of Amgen company is 23 amino acid of N-terminal (Δ 23hKGF) (Eric Hsu et al. of disappearance people keratinocyte growth factor I type, ProteinEngineering, Design ﹠amp; Selection vol.19 no.4 pp.147-153,2006), be used for the treatment of the serious oral mucosa lesion that neoplastic hematologic disorder patient high dose chemotherapy or combined radiotherapy cause by drugs approved by FDA in 2004.The Δ 23hKGF transformation period in vivo only is 2hr, and clinical administration dosage big (60 μ g/kg/ day) needs successive administration 7 days (1 time/day).
With in the research of pharmaceutical grade protein, it is numerous to solve short method of protein medicaments transformation period in body in treatment.Wherein one of effective means is that protein is become conjugate with the water-soluble polymers covalent attachment.And employed water-soluble polymers comprises polyoxyethylene glycol, poly lactose, polymeric amino acid etc., and wherein the use of polyoxyethylene glycol is the most general.
Polyoxyethylene glycol-protein conjugate has that good thermostability, resistance to enzymolysis, increase are water-soluble, transformation period in the extension body, reduce multiple advantage (Inada such as immunogenicity and toxicity, et al., J.Bioact.And CompatiblePolymers, 5:343 (1990); Delgalo, et al., Critical Reviews in Therapeutic Drug Carriersystems, 9:249 (1992); Katre, Advanced Drug Delivery Systems, 10:91 (1993)).First kind of medicine PEG-ADA listing of modifying with polyoxyethylene glycol (PEG) in 1991, Shang Shi product had PEG-Interferon, rabbit, PEG-granulocyte colony-stimulating factor, PEG-asparaginase, PEG-Somatostatin and PEG-ribavirin etc. in recent years.In addition, the protein drug that still has tens of kinds of PEG to modify is in research or clinical experiment stage.
Retrieving domestic and international patent and document finds, adopt the open source literature of polyethyleneglycol modified human keratinocyte Growth Factor that people (Zhifeng Huang.et al., Journal of Biotechnology 142 (2009) 242-249.) such as Zhifeng Huang polyethyleneglycol modified at the human keratinocyte Growth Factor N-terminal only arranged.This patent has been invented the free sulfhydryl groups of the 17th halfcystine of maleimide activatory mono methoxy polyethylene glycol specificity modification recombination deficient type human keratinocyte Growth Factor I type, this modifying method is modified the recombination deficient type human keratinocyte Growth Factor I type chemical coupling thing that forms, and has that decorating site is single-minded, a structure homogeneous, advantage such as easy to implement; What is more important can effectively be improved its internal metabolism dynamics under the biological activity that keeps human keratinocyte Growth Factor I type, realize prolong half-life, reduces administration frequency, and is more suitable as a kind of prevention or curative drug.
Summary of the invention
Covalently bound and the conjugate that forms of the free sulfhydryl groups of halfcystine that the objective of the invention is to adopt polyoxyethylene glycol and recombination deficient type human keratinocyte Growth Factor I type, this conjugate can effectively improve people's keratinocyte growth factor pharmacokinetics in vivo; This conjugate can be used as a kind of prevention or medicine simultaneously, or pharmaceutical composition, is used for the treatment of gastrointestinal mucosal damage that chemicotherapy is caused or ulcer mucositis, acute chemical damage at dose therapeutically effective.
Natural wild-type human keratinocyte Growth Factor is a single chain polypeptide that contains 163 amino-acid residues, contain and be positioned at 1,15, five halfcystines of 40,102,106, wherein Cys1-Cys15, Cys102-Cys106 form two pairs of disulfide linkage, Cys40 with the free sulfhydryl groups form exist (Timothy D.Osslund et al., prorein Science (1998), 7:1681-1690).
The free sulfhydryl groups of the halfcystine during the free sulfhydryl groups of the halfcystine of polyoxyethylene glycol of the present invention and recombination deficient type human keratinocyte Growth Factor I type is covalently bound, refer to the 17th halfcystine (Cys) of natural wild-type human keratinocyte Growth Factor I type (hKGF) N-terminal 23 amino acid whose human horny cell growth factor-2 mutants of disappearance (Δ 23hKGF), be equivalent to the 40th halfcystine of natural wild-type human keratinocyte Growth Factor I type.
In order to realize purpose of the present invention, the present invention has at first defined the aminoacid sequence or the composition of deletion human keratinocyte growth factor I type.Described deletion human keratinocyte growth factor I type is meant 23 the amino acid whose mutant of N-terminal disappearance in natural wild-type human keratinocyte Growth Factor I type, and sequence signature is (sequence is SEQ ID No:1):
Ser?Tyr?Asp?Tyr?Met?Glu?Gly?Gly?Asp?Ile?Arg?Val?Arg?Arg?Leu?Phe
Cys?Arg?Thr?Gln?Trp?Tyr?Leu?Arg?Ile?Asp?Lys?Arg?Gly?Lys?Val?Lys
Gly?Thr?Gln?Glu?Met?Lys?Asn?Asn?Tyr?Asn?Ile?Met?Glu?Ile?Arg?Thr
Val?Ala?Val?Gly?Ile?Val?Ala?Ile?Lys?Gly?Val?Glu?Ser?Glu?Phe?Tyr
Leu?Ala?Met?Asn?Lys?Glu?Gly?Lys?Leu?Tyr?Ala?Lys?Lys?Glu?Cys?Asn
Glu?Asp?Cys?Asn?Phe?Lys?Glu?Leu?Ile?Leu?Glu?Asn?His?Tyr?Asn?Thr
Tyr?Ala?Ser?Ala?Lys?Trp?Thr?His?Asn?Gly?Gly?Glu?Met?Phe?Val?Ala
Leu?Asn?Gln?Lys?Gly?Ile?Pro?Val?Arg?Gly?Lys?Lys?Thr?Lys?Lys?Glu
Gln?Lys?Thr?Ala?His?Phe?Leu?Pro?Met?Ala?Ile?Thr
Be included in simultaneously that N-terminal increases or reduce 1-5 amino acid whose varient on the basis of SEQ ID No:1 sequence.As increasing a methionine(Met) (Met) from SEQ ID No:1 sequence of N is terminal, or to increase by 5 aminoacid sequences be Pro-Glu-Arg-His-Thr-Arg or Met-Glu-Arg-His-Thr-Arg; Or reduce a Serine (Ser) from SEQ ID No:1 sequence of N is terminal, or to reduce by 5 aminoacid sequences be Ser-Tyr-Asp-Tyr-Met, or to reduce by 4 aminoacid sequences be Ser-Tyr-Asp-Tyr.
Described recombination deficient type human keratinocyte Growth Factor I type, be meant by the genetically engineered recombinant technology, recombinant expressed through intestinal bacteria, express after the chromatographic separation technology obtains purity greater than 95% recombination deficient type human keratinocyte Growth Factor I type (Δ 23hKGF).
In order to realize purpose of the present invention, the invention provides the polyethyleneglycol modified preparation of Δ 23hKGF.Described polyoxyethylene glycol is mono methoxy polyethylene glycol maleimide (Monomethoxy Polyethylene glycolMaleimide, mPEG-MAL) activated molecule, or be adjacent two thiopyridines (the MonomethoxyPolyethylene glycol Ortho-pyridyldisulfide of mono methoxy polyethylene glycol, mPEG-OPSS) activated molecule, or be mono methoxy polyethylene glycol vinyl sulphone (Monomethoxy Polyethylene glycol Vinylsufone, mPEG-VS) activated molecule, or mono methoxy polyethylene glycol sulfydryl (Monomethoxy Polyethylene glycol sulfhydryl, mPEG-SH) activated molecule, or be mono methoxy polyethylene glycol iodo-acetamide (Monomethoxy Polyethylene glycol Iodo acetamide, mPEG-IA) activated molecule, preferred mono methoxy polyethylene glycol-dimaleoyl imino activated molecule.Peg molecule can be straight chain type or ramiform, and molecular weight is between 5,000-40,000 dalton, wherein preferred 20,000 daltonian straight chain type peg molecules and 40,000 daltonian branched polyethylene glycol molecules.
Described Δ 23hKGF's is polyethyleneglycol modified, the Δ 23hKGF protein concentration that is meant the reorganization preparation is 1-10mg/ml, at the phosphate buffered saline buffer of 10-100mm, under the pH value 5.0-8.0, recombinant protein and polyoxyethylene glycol mass ratio are 1: the condition of 0.5-6 and mPEG-MAL, or mPEG-OPSS, or mPEG-SH, or mPEG-IA, through 4-37 ℃, behind the 100r/min stirring reaction 0.5-24hr, turn down the pH termination reaction.Adopt the chromatographic separation technology to remove not adorned Δ 23hKGF and free PEG molecule again, obtain the conjugate of purity greater than 95% Pegylation reorganization Δ 23hKGF.Wherein preferably modification condition is: Δ 23hKGF stoste 100mmol/L phosphate buffered saline buffer, the pH6.5 dilute sample is to 2mg/mL, by Δ 23hKGF and mPEG-MAL-20KD or with the mPEG-MAL-40KD mass ratio be that 1: 2 weighing m PEG-MAL-20KD or mPEG-MAL-40KD add in the Δ 23hKGF solution, 25 ℃, behind the 100r/min stirring reaction 2hr, turn down pH to 3.0 termination reaction with hydrochloric acid.This preparation method proves that through example the modification rate is high and modified specificity is single-minded, modifies the after product homogeneous, and is beneficial to enforcement.
The Pegylation recombination deficient type human keratinocyte Growth Factor I type (PEG-Δ 23KGF) of this patent invention technology preparation; in experimental animal; the acute liver damage that is caused by CC14 can be effectively protected in subcutaneous injection in 1 body, the acute mucous membrane of small intestine damage of protecting 5 Fluracils to cause.
Therefore, the chemical coupling thing of recombination deficient type human keratinocyte Growth Factor I type of the present invention, can be used as a kind of prevention or medicine, or pharmaceutical composition, under dose therapeutically effective, be used to prevent and treat gastrointestinal mucosal damage or ulcer mucositis and the acute chemical damage that chemicotherapy is caused.
So far the present invention is described in detail, by with reference to following example, can has more the present invention to be expressly understood, described example only is intended to limit the invention for the purpose of illustration and not.
Description of drawings
Fig. 1 recombinate that Δ 23hKGF expresses and purifying after the SDS-PAGE collection of illustrative plates, 1 for before intestinal bacteria induce among the A figure, 2 for after inducing; 1 is broken bacterium supernatant among the B figure, 2 be the SP column purification after, 3 be the heparin purifying after, M is the protein standard.
The purified SDS-PAGE collection of illustrative plates in before and after the mPEG-MAL-20k of Fig. 2 Δ 23hKGF modifies and modification back, wherein 1 for before modifying, and 2 for after the modification, and 3 for modifying the PEG-20K-Δ 23KGF of back purifying, and M is the protein standard.
Fig. 3 is before and after the mPEG-MAL-40k of Δ 23hKGF modifies and the purified SDS-PAGE collection of illustrative plates in modification back, and wherein 1 for before modifying, and 2 for after the modification, and 3 for modifying the PEG-20K-Δ 23KGF of back purifying, and M is the protein standard.
Embodiment
Example 1, the disappearance 23 amino acid whose human keratinocyte Growth Factor I types of N-terminal (Δ 23hKGF) recombinant expressed
According to Δ 23hKGF aminoacid sequence (SEQ ID No:1), the corresponding cDNA sequence of design intestinal bacteria preference codon comprises 5 ' end and 3 ' design limit restriction endonuclease Nde I and BamH I site (SEQ ID No:2) respectively.Entrust the full gene synthetic of precious biotechnology (Dalian) company limited, and embed pUC18 carrier and called after pUC18-Δ 23hKGF/DH5 α.
Plasmid pUC18-Δ 23hKGF DH5 α and expression vector plasmid pET-3c restriction enzyme Nde I and BamH I double digestion, according to 1% agarose electrophoresis result, reclaim the pET-3c enzyme and cut the fragment of the about 4638bp in back, reclaim the fragment that pUC18-Δ 23hKGF/DH5 α enzyme is cut the about 450bp in back, two fragments are connected with the T4 ligase enzyme, with LA (Tryptones 2g, yeast extract 1g, NaCl 2g, agar 3g, it is fixed molten to 200mL to add water, 121 ℃, autoclaving 30min, when temperature drops to 50 ℃ of left and right sides, add Amp to final concentration be 100ug/mL, get an amount of culture dish that falls, solidify the back the LA agar plate) the plate screening recon, Nde I and BamH I double digestion are identified, positive colony.Correct plasmid called after pET-3c-Δ 23hKGF.Again plasmid pET-3c-Δ 23hKGF is transformed in BL21 (DE3) the plysS host bacterium correct engineering bacteria name pET-3c-Δ 23hKGF/BL21 (DE3) plysS that expresses of screening.Carrier that is used to express and host bacterium bacterium are purchased the company in Novagen, and Nde I and BamH I enzyme are purchased in precious biotechnology (Dalian) company limited.
With the best engineering strain streak inoculation of above-mentioned expression in the LA agar plate, 37 ℃ of overnight incubation.The picking lawn is inoculated in and contains LB liquid nutrient medium (Tryptones 10g from the LA flat board of incubated overnight, yeast extract 5g, NaCl 10g, it is fixed molten to 1000mL to add water, 121 ℃, autoclaving 30min gets final product) in the test tube, cultivated 12 hours for 37 ℃, be transferred in the 1000mL triangular flask that contains 200mL LB nutrient solution in 1% ratio then, 37 ℃ of overnight incubation promptly become and go up a jar seed liquor.Last jar seed liquor is inoculated in the 30L fermentor tank that contains 20L M9YT nutrient solution in 5% ratio, 37 ℃ of cultivations, have suffered in the fermenting process, keep dissolved oxygen more than 30% by adjusting rotating speed, air capacity, pure oxygen amount, the ammoniacal liquor with 28% is regulated pH and is also remained on 7.0.To bacterium liquid OD600=10～14 o'clock, adding final concentration was the IPTG of 0.1mmol/L, and 28 ℃ are continued to cultivate and stop fermentation (Figure 1A) after 6 hours, collect bacterium liquid, and centrifugal 10 minutes of 8000rpm abandons supernatant, collected thalline and put into-20 ℃ of refrigerators and preserve standby.
The purifying preparation of example 2, Δ 23hKGF
Take out thalline from-20 ℃ of refrigerators, by 1: 15w/v puts into bacterial lysate (50mmol/L Tris-HCl, 5mmol/LEDTA, 10 μ g/mlDNase, 2mm MgCl
2, pH 8.5-9.0) in, 37 ℃ are stirred down and treat broken that bacterium liquid does not add the trisodium citrate of final concentration 0.1M during thickness, continues stirring 0.5h.Regulating pH is 7.5, and 10000rpm is centrifugal, and 15min collects supernatant.(balance liquid: 50mmpb, pH7.5), multiple equilibria is used the 50-500mmNacl linear gradient elution to SP-Sepharose Fast Flow post on the supernatant liquor, collects elution peak, detects through SDS-PAGE, determines to contain the proteic elution peak of main purpose.(balance liquid: 20mm pb, pH7.5), multiple equilibria with 50-500mm Nacl linear gradient elution, is collected elution peak to Heparin post on the purpose sample of SP wash-out, detects through SDS-PAGE and HPLC, obtains purity greater than 95% Δ 23hKGF (Figure 1B).
The mPEG-MAL-20K of example 3, Δ 23hKGF modifies and modifies the purifying of back (PEG-20K-Δ 23KGF)
Purity is greater than 95% Δ 23hKGF solution 100mmol/L phosphate buffered saline buffer, pH6.5, dilute sample is to 2mg/mL, press Δ 23hKGF and mPEG-MAL-20KD (straight chain type, purchase in the strong triumphant Science and Technology Ltd. in Beijing) mass ratio is that 1: 2 weighing m PEG-MAL-20KD adds in the Δ 23hKGF solution, 25 ℃, 100r/min stirring reaction 2hr turns down the pH termination reaction with hydrochloric acid.Δ 23hKGF after the modification (PEG-20K-Δ 23KGF) solution dialysed overnight (dialyzate: 20mmNaAc, pH5.0), SP-Sepharose Fast Flow post (balance liquid: 20mmNaAc on the PEG-20K-Δ 23KGF solution after the dialysis, pH5.0), multiple equilibria with 50-500mm Nacl linear gradient elution, is collected elution peak, detect through SDS-PAGE and HPLC, obtain purity greater than 95% PEG-20K-Δ 23KGF (Fig. 2).
The mPEG-MAL-40K of example 4, Δ 23hKGF modifies and modifies the purifying of back (PEG-40K-Δ 23KGF)
Purity is greater than 95% Δ 23hKGF solution 100mmol/L phosphate buffered saline buffer, pH6.5, dilute sample is to 2mg/mL, by Δ 23hKGF and mPEG-MAL-40KD (ramiform) mass ratio is that 1: 2 weighing m PEG-MAL-40KD adds in the Δ 23hKGF solution, 25 ℃, behind the 100r/min stirring reaction 2hr, turn down pH to 3.0 termination reaction with hydrochloric acid.Δ 23hKGF after the modification (PEG-40K-Δ 23KGF) solution dialysed overnight (dialyzate: 20mmNaAc, pH5.0), SP-Sepharose Fast Flow post (balance liquid: 20mmNaAc on the PEG-40K-Δ 23KGF solution after the dialysis, pH5.0), multiple equilibria with 50-500mm Nacl linear gradient elution, is collected elution peak, detect through SDS-PAGE and HPLC, obtain purity greater than 95% PEG-40K-Δ 23KGF (Fig. 3).
The external activity of example 5, Δ 23hKGF, PEG-20K-Δ 23KGF and PEG-40K-Δ 23KGF detects
Sample Δ 23hKGF, PEG-20K-Δ 23KGF and PEG-40K-Δ 23KGF are diluted to 1 μ g/ml with 1640 substratum that contain 0.5% foetal calf serum respectively, again by 7 concentration of 1: 4 gradient dilution.With the every hole 3 * 10 of NBL-7 cell
3Be inoculated in 96 porocyte culture plates, place 37 ℃, 5%CO
2Overnight incubation under the condition, multiple hole adds the sample of the different weaker concns of above preparation behind the exhaustion supernatant.Place CO2gas incubator continue to cultivate 72h, every hole adds MTT 10 μ l dyeing, discards nutrient solution after cultivating 4～6h again, adds the DMSO of 100 μ l, measures each hole absorbancy OD value behind the vibration mixing under the 570nm wavelength.International active standard substance (deriving from NIBSC) according to KGF-I calculate the external biological specific activity (IU/mg) that PEG modifies the Δ 23hKGF of front and back.
The result:
| Group | EC50(ng/ml) | Than live (IU/mg) |
| ?Δ23hKGF | 25.1 | 1.14×10 6 |
| ?PEG-20K-Δ23KGF | 37.8 | 0.75×10 6 |
| ?PEG-40K-Δ23KGF | 64.2 | 0.42×10 6 |
Measure the EC of Δ 23hKGF, PEG-20K-Δ 23KGF and PEG-40K-Δ 23KGF through the NBL-7 cytoactive
50Be respectively 25.1,37.8 and 64.2ng/ml.The specific activity that the active standard substance of measuring down through the same terms of KGF-I calculate is respectively 1.14 * 10
6, 0.75 * 10
6With 0.42 * 10
6IU/mg.The activity of the Δ 23hKGF of proof after PEG modifies has kept between the 30-60%, and wherein the decorating molecule amount is big more, and retentive activity is low more.
Example 5, Δ 23hKGF, PEG-20K-Δ 23KGF and PEG-40K-Δ 23KGF are in the research of the intravital pharmacokinetics of new zealand rabbit
30 of new zealand white rabbits are divided into three groups (Δ 23hKGF group, PEG-20K-Δ 23KGF and PEG-40K-Δ 23KGF groups) at random.Three groups all by the 4.0mg/kg subcutaneous injection; Get blood in injection back 0,30min, 60min, 1hr, 2hr, 4hr, 4hr, 24hr, 48hr, 72hr, 96hr, 120hr, 144hr from ear vein.Adopt the ELISA Kit (R﹠amp of rhKGF; D company) measures corresponding Plasma Concentration, with change of pharmacokinetics in the body behind standard substance curve calculation Δ 23hKGF, PEG-20K-Δ 23KGF and the PEG-40K-Δ 23hKGF single-dose.
Experimental result shows, the transformation period t of Δ 23hKGF
α 1/2Be 3.9hr, t
β 1/2Be 6.2hr; And the transformation period t of PEG-20K-Δ 23KGF
α 1/2Be 57.4hr, t
β 1/2Be 154.9hr; And the transformation period t of PEG-40K-Δ 23KGF
α 1/2Be 61.2hr, t
β 1/2Be 181.5hr.The result proves the interior transformation period significant prolongation of the body of PEGization Δ 23hKGF.
Example 6, Δ 23hKGF and PEG-20K-Δ 23KGF are to the therapeutic evaluation of model mice CC14 acute liver damage
40 of kunming mices, 20-25g/, male and female half and half are divided into 4 groups (normal group, model control group, Δ 23hKGF group and PEG-20K-Δ 23KGF groups) at random.A normal group injecting normal saline, other three groups of mouse are pressed 150mg/kg abdominal cavity CC14 solution 1 time, and control group is being injected CC14 the day before yesterday, and subcutaneous injection physiological saline 1 time/day, was injected 3 days continuously; Δ 23hKGF group is being injected CC14 the day before yesterday, and the subcutaneous 4.0mg/kg injection Δ 23hKGF that presses 1 time/day, injected 3 days continuously.PEG-20K-Δ 23KGF group is being injected CC14 the day before yesterday, and the subcutaneous 4.0mg/kg injection PEG-20K-Δ 23KGF that presses only injects 1 time.Eyeball of mouse is got blood behind CC14 injection 48h, gets determination of serum ALT and AST unit of activity after centrifugal.
The result:
| Group | ALT (Unit/L) (mean value) | AST (Unit/L) (mean value) |
| Normally | 32.1 | 20.9 |
| The model contrast | 347.8 | 294.5 |
| Δ23hKGF | 246.9 | 179.5 |
| PEG-20K-Δ23KGF | 84.3 | 101.7 |
Last watch test result shows: Δ 23hKGF can reduce continuously the rising of ALT and AST in the blood that the liver injury that caused by CC14 causes after the injection, its reduced rate is 30-40%, and only once PEG-20K-Δ 23KGF of injection can reduce under same dose that ALT and AST raise in the blood that the liver injury that caused by CC14 causes, its reduced rate is 300-400%, is ten times of Δ 23KGF.
Example 7, Δ 23hKGF and PEG-20K-Δ 23KGF are to the therapeutic evaluation of model mice 5 Fluracil intestinal mucosal injuries
60 of kunming mices, 20-25g/, male and female half and half are divided into 4 groups (normal group, model control group, Δ 23hKGF group and PEG-20K-Δ 23KGF) at random.A normal group injecting normal saline, other three groups of mouse are pressed 50mg/kg/ days abdominal injection 5 Fluracils, inject continuously 5 days.Control group is in the first two day beginning of injection 5 Fluracils, and subcutaneous injection physiological saline 1 time/day, was injected 10 days continuously; Δ 23hKGF group is in the first two day beginning of injection 5 Fluracils, and the subcutaneous 4.0mg/kg injection Δ 23hKGF that presses 1 time/day, injected 10 days continuously; PEG-20K-Δ 23KGF group is in the first two day beginning of injection 5 Fluracils, and the subcutaneous 4.0mg/kg injection PEG-20K-Δ 23hKGF that presses 1 time/3 days, injects 3 times continuously.Experimental session is investigated body weight change, the mortality ratio of each group, dissects animal simultaneously, observes intestinal tract injury cardinal principle and pathological change.
The result:
As seen from the above table, the intact animal treated animal is all survived, and body weight increases gradually, and naked eyes sight mucous membrane of small intestine does not have congested and necrosis, nothing come off the no abnormal change of pathology.And model control group lost weight since the 5th day, animal dead occurred by 10 days, and mortality ratio is that 26.7%, 15 day mortality ratio is 46.7%, and the visible serious big area hyperemia of small intestine is with necrosis, attenuation and come off the visible down inflammatory cell infiltrations in a large number of mirror.Δ 23hKGF and PEG-20K-Δ 23KGF treated animal body weight to 15 day still have slight increase, and during to 15 days, mucous membrane of small intestine damage and pathological change that mortality ratio is respectively 20% and 6.7%, two treated animal all significantly are lighter than model control group.
SEQUENCE?LISTING
＜110〉Chongqing Fujin Biological Medicine Co. Ltd
＜120〉the chemical coupling thing of recombination deficient type human keratinocyte Growth Factor I type
<130>
<160>2
<170>PatentIn?version?3.3
<210>1
<211>140
<212>PRT
＜213〉artificial sequence
<400>1
Ser?Tyr?Asp?Tyr?Met?Glu?Gly?Gly?Asp?Ile?Arg?Val?Arg?Arg?Leu?Phe
1 5 10 15
Cys?Arg?Thr?Gln?Trp?Tyr?Leu?Arg?Ile?Asp?Lys?Arg?Gly?Lys?Val?Lys
20 25 30
Gly?Thr?Gln?Glu?Met?Lys?Asn?Asn?Tyr?Asn?Ile?Met?Glu?Ile?Arg?Thr
35 40 45
Val?Ala?Val?Gly?Ile?Val?Ala?Ile?Lys?Gly?Val?Glu?Ser?Glu?Phe?Tyr
50 55 60
Leu?Ala?Met?Asn?Lys?Glu?Gly?Lys?Leu?Tyr?Ala?Lys?Lys?Glu?Cys?Asn
65 70 75 80
Glu?Asp?Cys?Asn?Phe?Lys?Glu?Leu?Ile?Leu?Glu?Asn?His?Tyr?Asn?Thr
85 90 95
Tyr?Ala?Ser?Ala?Lys?Trp?Thr?His?Asn?Gly?Gly?Glu?Met?Phe?Val?Ala
100 105 110
Leu?Asn?Gln?Lys?Gly?Ile?Pro?Val?Arg?Gly?Lys?Lys?Thr?Lys?Lys?Glu
115 120 125
Gln?Lys?Thr?Ala?His?Phe?Leu?Pro?Met?Ala?Ile?Thr
130 135 140
<210>2
<211>439
<212>DNA
＜213〉artificial sequence
<400>2
cgcatatgtc?ttacgactac?atggaaggcg?gcgacatccg?tgttcgtcgt?ctgttctgcc 60
gtacccagtg?gtacctgcgt?atcgacaaac?gtggcaaagt?taaaggcacc?caggaaatga 120
aaaacaacta?caacatcatg?gaaatccgta?ccgttgctgt?tggcatcgtt?gctatcaaag 180
gcgttgaatc?tgaattctac?ctggctatga?acaaagaagg?caaactgtac?gctaaaaaag 240
aatgcaacga?agactgcaac?ttcaaagaac?tgatcctgga?aaaccactac?aacacctacg 300
cttctgctaa?atggacccac?aacggcggcg?aaatgttcgt?tgctctgaac?cagaaaggca 360
tcccggttcg?tggcaaaaaa?accaaaaaag?aacagaaaac?cgctcacttc?ctgccgatgg 420
ctatcacctt?aggatccgc 439
Claims (8)
1. the present invention is the conjugate that the peg molecule chemically modified to the free sulfhydryl groups of the halfcystine of reorganization deletion human keratinocyte growth factor I type forms.
2. the described recombination deficient type of claim 1 human keratinocyte Growth Factor I type refers to the natural human keratinocyte Growth Factor I type that contains 163 amino acid compositions at 23 amino acid of N-terminal disappearance, and aminoacid sequence is SEQ ID No:1.Be included in simultaneously that N-terminal increases or reduce 1-5 amino acid whose varient on the basis of SEQ ID No:1 sequence.
3. the free sulfhydryl groups of the halfcystine of the described recombination deficient type of claim 1 human keratinocyte Growth Factor I type refers to be arranged in the 17th amino acids of aminoacid sequence SEQ ID No:1.
4. the described peg molecule of claim 1, it is characterized in that, polyoxyethylene glycol is mono methoxy polyethylene glycol maleimide (Monomethoxy Polyethylene glycol Maleimide, mPEG-MAL) activated molecule, or be adjacent two thiopyridines (the Monomethoxy Polyethylene glycolOrtho-pyridyldisulfide of mono methoxy polyethylene glycol, mPEG-OPSS) activated molecule, or be mono methoxy polyethylene glycol vinyl sulphone (Monomethoxy Polyethylene glycol Vinylsufone, mPEG-VS) activated molecule, or mono methoxy polyethylene glycol sulfydryl (Monomethoxy Polyethylene glycol sulfhydryl, mPEG-SH) activated molecule, or be mono methoxy polyethylene glycol iodo-acetamide (Monomethoxy Polyethylene glycol Iodo acetamide, mPEG-IA) activated molecule, preferred mono methoxy polyethylene glycol maleimide activated molecule.
5. claim 1,4 each described peg molecules is characterized in that the polyglycol molecule can be straight chain type or ramiform;
6. claim 1,4,5 each described peg molecules is characterized in that the peg molecule molecular weight is 5,000-40,000 dalton, wherein preferred 20,000 daltonian straight chain type peg molecules and 40,000 daltonian branched polyethylene glycol molecules.
7. conjugate according to claim 1 can be used as a kind of prevention and curative drug, it is characterized in that, described medicine is the albumen of the Pegylation reorganization recombination deficient type human keratinocyte Growth Factor I type of treatment effective dose.
8. claim 7 is described as the prevention and the conjugate of medicine, and the gastrointestinal mucosal that can be used for preventing and treat chemicotherapy to cause damages, the clinical treatment of acute chemical damage or ulcer mucositis.
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| CN110183529A (en) * | 2019-05-23 | 2019-08-30 | 重庆派金生物科技有限公司 | A kind of recombination and preparation of deletion human keratinocyte growth factor-1 |
| CN110183529B (en) * | 2019-05-23 | 2023-06-02 | 重庆派金生物科技有限公司 | Recombinant preparation method of deletion type human keratinocyte growth factor-1 |
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