CN100356981C - Ligand oligopeptide specific combining human epidermal growth factor receptor EGFR - Google Patents
Ligand oligopeptide specific combining human epidermal growth factor receptor EGFR Download PDFInfo
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- CN100356981C CN100356981C CNB2004100173346A CN200410017334A CN100356981C CN 100356981 C CN100356981 C CN 100356981C CN B2004100173346 A CNB2004100173346 A CN B2004100173346A CN 200410017334 A CN200410017334 A CN 200410017334A CN 100356981 C CN100356981 C CN 100356981C
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Abstract
The present invention provides a gene transfer system mediated by an epidermal growth factor receptor (EGFR). The system comprises (a) ligand oligopeptide specifically combined in the EGFR, (b) polycation polypeptide or protein and (c) optional endocytic corpuscular release oligopeptide and (d) exogenous DNA. The gene transfer system is characterized in that exogenous genes can be effectively target-introduced into tumor cells expressing the EGFR in vivo and in video so as to inhibit the growth of the tumor cells.
Description
Technical field
The present invention relates to molecular biology and medical domain.Specifically, the present invention relates to the part oligopeptide of the efficient specific bond human epidermal growth factor acceptor of class EGFR and the DNA sequence of this polypeptide of encoding.The invention still further relates to the purposes of this DNA and part oligopeptide.
Background technology
The mechanism of tumor development mainly comes from the activation of tumor gene (oncogene) or the inactivation of CDKN2.EGFR is as the former tumor gene of a wide expression, and its change (disappearance, high expressed etc.) plays an important role in the generation evolution of tumor.
EGFR is a single transmembrane receptor, is made up of 1186 amino acid residues, and molecular weight is 170kD, has an extracellular region, strides film district and an intracellular region.Extracellular region is receptor and the bonded zone of native ligand (comprising EGF, TGF-α and amphiregulin (Amphiregulin)).Intracellular region has tyrosine kinase district and carboxyl terminal district.The carboxyl terminal district can with downstream joint protein binding, activate the downstream signal pathway.
EGFR family also has erbB2, erbB3 and erbB4 except EGFR.The tyrosine kinase district of these four receptors is very conservative, but carboxyl terminal region sequence conservative is low, so the activated downstream signal path of EGFR family is very complicated.When the EGFR receptor changes with making receptor conformation after native ligand combines, homodimerization occurs or form heterodimerization with its family member, and then activation intracellular region tyrosine kinase activity latter phosphorylation carboxyl terminal district, produce the signal conduction by the joint albumen and the downstream factor, make gene activation, expression impels cell growth and mitotic albumen, thereby makes cell growth, propagation.Because at normal cell, the expression of EGFR is controlled, therefore can't produce harmful effect by pair cell.In some cases, the EGFR abnormal expression, as extracellular part disappearance, will make the EGFR kinase activity not rely on part to activate lastingly, thereby cause cell malignant proliferation to occur.
Except deletant, EGFR has high expressed in tumors such as tumor colli, nonsmall-cell lung cancer.Therefore, had a lot of researchs EGFR as the tumor treatment target spot.Comprise anti-egfr antibodies, be receptor-mediated gene therapy vector system with EGFR.
PCT application PCT/CN97/00106 (WO98/18951) discloses the novel receptor-mediated gene transfer system that is used for the targeting therapy of tumor, wherein discloses to comprise at EGFR, IGF I/II R and three kinds of receptor-mediated gene transfer systems of VEGFR and be used for oncotherapy.
Yet, at present be that native ligand such as the EGF bullet as carrier system is adopted in the gene therapy of receptor mostly with EGFR.Split source activity because EGF has stronger silk, can promote cell mitogen and angiogenesis, and molecular weight is big, can be to the active considerable influence that produces of other elements of carrier system, the part oligopeptide of therefore seeking EGFR seems particularly important.In addition, though a kind of known GE7 part oligopeptide also has certain affinity with EGFR, iodine shows that in conjunction with experiment the affinity of this polypeptide and EGFR receptor is not high.
Therefore, this area presses for the part oligopeptide of the new targeting EGFR of exploitation and based on the gene transfer system of the targeting therapy of tumor of EGFR part oligopeptide.Gene therapy and other targeted therapy measures by targeting EGFR reach the purpose for the treatment of disease.
Summary of the invention
The objective of the invention is just to provide the part oligopeptide of a kind of EGFR, part oligopeptide of the present invention can combine with the EGFR specificity, and by receptor mediated endocytosis, can be used as the targeting peptide is used for the targeting gene transfer system on the one hand; Can connect the tumor cell of the direct targeting EGFR receptor of radionuclide, chemicals and biotoxin high expressed on the other hand, thereby reach the purpose of targeted therapy tumor.
Another object of the present invention provided based on the gene transfer system of the bonded part oligopeptide of EGF-R ELISA EGFR.This transfer system imports the tumor cell of expressing the EGFR receptor by receptor mediated endocytosis with foreign DNA targeting ground, thereby reaches the purpose of treatment tumor.
In a first aspect of the present invention, a kind of gene transfer system of targeting gene therapy of EGF-R ELISA EGFR mediation is provided, comprise the part oligopeptide that (a) specificity is incorporated into the EGFR receptor, wherein said part oligopeptide contains SEQ ID NO:1,2 or 3 aminoacid sequence; (b) polycation polypeptide or albumen.
In another preference, described gene transfer system also contains (c) endocytosis corpusculum and discharges oligopeptide.
In another preference, described gene transfer system also contains (d) foreign DNA.
In another preference, the aminoacid sequence of described part oligopeptide is SEQ ID NO:1,2 or 3.
In another preference, the polycation polypeptide is selected from down group: poly-D-lysine, poly-acetimide, protamine, histone, adenovirus pV albumen, adenovirus pVII albumen, m μ albumen, and through polyethyleneglycol modified above-mentioned albumen and composition thereof.
In another preference, described foreign DNA is selected from down group: the recombinant eukaryotic expression plasmid DNA of antioncogene, apoptosis gene, cytokine gene, oncogene antisense sequences, and composition thereof.
In another preference, described foreign DNA is selected from down group:
(i) antioncogene: p53, Rb;
(ii) apoptosis gene: Caspase, p15, p16 and p21
WAF-1
(iii) cytokine gene: GM-CSF gene, TNF α gene, INF α, γ gene, IL2, IL3, IL4, IL12, IL15, IL18 gene;
The (iv) antisense sequences of oncogene: the antisense sequences of oncogene ras, c-myc, Akt;
(v) recombinant eukaryotic expression plasmid: viral recombinant eukaryotic expression plasmid and be the non-viral recombinant eukaryotic expression plasmid of cis-regulating element with SV40, CMV promoter.
In another preference, described component (a) part oligopeptide, (b) polycation polypeptide and (c) both or the three that discharge in the oligopeptide of endocytosis corpusculum exist with the fusion rotein form.
In another preference, described endocytosis corpusculum discharges the oligopeptide element and is selected from down group: HA20 or VP-1.
In a second aspect of the present invention, a kind of oligopeptide (length is less than 20 aminoacid) is provided, it is incorporated into the EGFR receptor, contains following aminoacid sequence: SEQ ID NO:1,2 or 3.
In a third aspect of the present invention, a kind of method of gene transfer system of the targeting gene therapy for preparing EGF-R ELISA EGFR mediation is provided, comprise step: the part oligopeptide, (b) polycation polypeptide or the albumen that (a) specificity are incorporated into the EGFR receptor discharge oligopeptide with optional (c) endocytosis corpusculum and mix, and form mixture.Preferably, for component (a) and (b), (c), can use any the two formation binary complex.
In a fourth aspect of the present invention, a kind of conjugate is provided, it contain the above-mentioned part oligopeptide of the present invention and with the link coupled radionuclide of described part oligopeptide, chemicals and biotoxin.These conjugates can directly apply to the treatment malignant tumor relevant with the EGFR overexpression, as nonsmall-cell lung cancer, tumor of head and neck.
In a fifth aspect of the present invention, a kind of pharmaceutical composition is provided, it contains the part oligopeptide of the present invention of safe and effective dosage, and pharmaceutically acceptable excipient, diluent or carrier.Pharmaceutical composition of the present invention can be used for treating the malignant tumor relevant with the EGFR overexpression, as nonsmall-cell lung cancer, tumor of head and neck.
In a sixth aspect of the present invention, the purposes of oligopeptide of the present invention is provided, they are used to prepare the detectable that detects EGFR or are used to prepare medicine.
Description of drawings
Fig. 1 is polypeptide GE9 mass spectral analysis figure.
Fig. 2 is
125I labelling GE9 combines the result of the test sketch map with SMMG7721 cell surface EGFR receptor-specific.
Fig. 3 is
125I labelling GE9 combines the result of the test sketch map with the EGFR protein-specific.
Fig. 4 is
125I labelling GE9 combines the dissociation constant curve with the EGFR receptor-specific.
Fig. 5 is phage clone and cell surface EGFR receptor-specific binding immunoassay group result.
Fig. 6 has shown
125I labelling GE11 combines experimental result with the EGFR protein-specific.
Fig. 7 has shown phage recovery test result.
The specific embodiment
The inventor is through extensive and deep research, prepared that specificity is incorporated into the part oligopeptide of EGFR receptor and based on the gene transfer system of this part oligopeptide.GE9 (SEQ ID NO:1), GE10 (SEQ IDNO:2) and GE11 (SEQ ID NO:3) are by the phage peptide library triage techniques, directly obtain from the peptide storehouse and the bonded polypeptide of EGFR acceptor molecule.The ad-hoc location of the specific amino acids of these polypeptide is avtive spots of polypeptide ligand and EGFR receptors bind, and EGFR acceptor molecule polypeptide ligand motif has just determined to combine with EGFR the living features of polypeptide.
Utilize the characteristic of part oligopeptide identification EGFR receptor of the present invention, endocytosis through cell, by this carrier system foreign DNA is optionally imported the tumor cell of expressing the EGFR receptor, thereby can suppress growth of tumour cell or kill tumor cell, reach the purpose of treatment tumor.
The part oligopeptide
The part oligopeptide is the key component of targeting non-virus carrier of the present invention.As used herein, " part oligopeptide (Ligand oligopeptide, LOP) " and " receptor identification oligopeptide " is used interchangeably, and refers to any identification and is incorporated into the oligopeptide that the surface contains the target cell of EGFR receptor.As mentioned above, but part oligopeptide specific recognition of the present invention and be incorporated into the EGFR receptor.Particularly, part oligopeptide of the present invention is meant and the bonded polypeptide of surface of cell membrane protein receptor EGFR extracellular region, perhaps with the bonded polypeptide of soluble EGFR molecule, comprises pattern of fusion polypeptide and free type polypeptide.
Ligand polypeptide of the present invention also comprises variant form identical function, SEQ IDNO.1-3 sequence that has with the EGFR receptors bind.These variant forms comprise (but being not limited to): several (are generally 1-5, preferably 1-3, more preferably 1-2,1 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) aminoacid.
One skilled in the art will appreciate that nonpolar (hydrophobicity) aminoacid comprises alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.Polar neutral amino acid comprises glycine, serine, threonine, tyrosine, agedoite and glutamine.Basic amino acid comprises lysine, histidine and arginine.Acidic amino acid comprises glutamic acid and aspartic acid.In the art, when replacing, can not change the function of polypeptide usually with the close or similar aminoacid of performance.In addition, add one or several aminoacid at C-terminal and/or N-terminal and also can not change proteinic function usually.
As used herein, term " part oligopeptide " comprise can with active fragment and the reactive derivative of the bonded GE9 of EGFR, GE10 and GE11.Also comprise the fusion rotein or the chimeric protein that form with function or character known protein.
As used herein, term " part oligopeptide " also comprises the polypeptide of amide or ester or its salt form.Both comprised free type polypeptide, pattern of fusion polypeptide and mosaic type polypeptide, comprised also that with polypeptide be monomeric various polymer.For example, contain protein molecule, chimeric protein molecule or polymers a kind of among SEQ ID NO:1, SEQ ID NO:2 and the SEQ ID NO:3, two kinds or three seed amino acid sequences.
In the application's description and accompanying drawing, be used to represent that base, amino acid whose abbreviation are to be recommended by biochemistry name IUPCA-IUB committee.Wherein aminoacid is generally the L type, unless other explanation is arranged.Polypeptide described in this description, by convention, left distal end is amino terminal (a N-end), right end is carboxyl terminal (a C-end).Usually when the C-end be carboxyl (COOH) or carboxylate (COO-) time, it can be amide (COONH2) or ester (COOR) form.Ester residue R comprises C1-6 alkyl (as methyl, ethyl, n-propyl group, isopropyl, n-butyl etc.), C3-8 cycloalkyl (as cyclopenta, cyclohexyl etc.), C6-12 aryl (as phenyl, a-naphthyl etc.), C7-14 alkyl (as benzyl, phenethyl etc.).
The particularly preferred part oligopeptide of one class is that length is 9-20 aminoacid, and contains the oligopeptide of the elementary cell sequence that is selected from down group:
CKSPEPQHC(SEQ?ID?NO：1)；
LHLWVPEPWTQT(SEQ?ID?NO：2)；
YHWYGYTPQNVI(SEQ?ID?NO：3)。
More preferred example is following oligopeptide:
Oligopeptide GE9, its aminoacid sequence are CKSPEPQHC (SFQ ID NO:1);
Oligopeptide GE10, its aminoacid sequence are LHLWVPEPWTQT (SEQ ID NO:2);
Oligopeptide GE11, its aminoacid sequence are YHWYGYTPQNVI (SEQ ID NO:3);
In addition, part oligopeptide of the present invention comprises that also the aminoacid in one or several (1-8 usually, preferably 1-5) site can replace formed part oligopeptide with the aminoacid of similar performance.
Part oligopeptide of the present invention can contain single elementary cell sequence, or a plurality of (as 2-10) elementary cell sequence.
Part oligopeptide of the present invention can be easily with synthetic or genetic engineering recombinant methods.About these synthetic oligopeptide method for makings, can be referring to such as WO98/18951 many documents such as (PCT/CN97/00106).
The present invention also provides the coded sequence of EGFR part oligopeptide such as GE9, GE10, GE11, and they contain the nucleotide sequence shown in the SEQ ID NO:5,6 and 7 respectively.These coded sequences can obtain with conventional artificial synthesis or recombination method.
The present invention also provides a kind of conjugate, it contain the above-mentioned part oligopeptide of the present invention and with the link coupled radionuclide of described part oligopeptide, chemicals and biotoxin.These conjugates can directly apply to the treatment malignant tumor relevant with the EGFR overexpression, as nonsmall-cell lung cancer, tumor of head and neck.
The polycation polypeptide
The polycation polypeptide be targeting non-virus carrier of the present invention another key component.
As used herein, " polycation polypeptide " refers to that a class is rich in the polypeptide of basic amino acid, and its basic amino acid (lysine, histidine and arginine) ratio is about more than 20%, thereby positively charged, can combine with electronegative DNA mat electrostatic attraction, make DNA more stable.
Can be used for polycation polypeptide of the present invention and be not particularly limited, as long as its basic amino acid (lysine and arginine) ratio is about more than 20%, preferably 30%, more preferably more than 40%, thereby positively charged getting final product.Representational example comprises: poly-D-lysine, poly-acetimide, protamine, histone, adenovirus pV albumen, adenovirus pVII albumen, M μ albumen, above-mentioned albumen of modifying through Polyethylene Glycol (PEG) and composition thereof.
The endocytosis corpusculum discharges the oligopeptide element
Targeting non-virus carrier of the present invention also can contain the endocytosis corpusculum and discharge the oligopeptide element, swallows the effect that the DNA of cell is degraded in it plays and prevents.
Any endocytosis corpusculum discharges oligopeptide and all can be used for the present invention, and representational example comprises (but being not limited to): HA20, VP-1.
The targeting non-virus carrier
As used herein, " targeting non-virus carrier " of the present invention refers to contain following component (a) and (b) or contain component (a) and (b) and mixture (c) or complex: (a) the specificity part oligopeptide, (b) polycation polypeptide, (c) endocytosis corpusculum that are incorporated into the EGFR receptor discharges oligopeptide.
In the present invention, component (a) and (b), (c) can be non-types of attachment, also can be any both or three link together by mode covalently or non-covalently.Particularly preferred form comprises: part oligopeptide-polycation polypeptide binary complex, polycation polypeptide-endocytosis corpusculum discharge the oligopeptide binary complex, part oligopeptide-polycation polypeptide-endocytosis corpusculum discharges the oligopeptide ternary complex.For example HA20-poly-D-lysine, HA20-protamine, the covalently bound thing of HA20-histone etc.
Connection can be passed through chemical method (as coupling agents such as use SPDP), also can pass through recombination method, for example forms fusion rotein.
For fusion rotein, at (a) part oligopeptide, (b) polycation polypeptide, and (c) the endocytosis corpusculum discharges between the oligopeptide element, can randomly contain connection peptides, so that increase the flexibility and the stretching of complex polypeptide space structure, allow each element performance function separately.Can be used for connection peptides of the present invention and be not particularly limited, needed only interconnect function.One class connection peptides example is (Gly)
2-6Ser, for example (Gly)
4Ser.Connection peptides can be used in a plurality of series connection.
(a) of the present invention part oligopeptide, (b) the polycation polypeptide and (c) the endocytosis corpusculum discharge the oligopeptide element can be with the amino acid replacement of similar performance, as long as still keep separately similarly biological function.
The preparation method of targeting non-virus carrier
Usually, above-mentioned (a) and (b), (c) component or its complex are mixed, can obtain targeting non-virus carrier of the present invention.Wherein, the mixed proportion of (a) and (b), (c) component is generally 0.5-1: 1-2: 0-1 preferably is 0.8-1: 1-1.5: 0.5-1.
In addition, when with (a)-(b) complex with (b)-(c) when the form of complex is used, (a)-(b) complex: (b)-(c) mixed proportion of complex is generally 0.8-1.2: 0.8-1.2 preferably is 0.9-1.1: 0.9: 1.1.
Foreign DNA
Can be used for foreign DNA of the present invention is not particularly limited, can be various therapeutic or preventative DNA, for example genes of interest, antisense oncogene, antioncogene, suicide gene, natural death of cerebral cells gene, cytokine gene or its combination perhaps contains the carrier for expression of eukaryon DNA of said gene.The proto-oncogene antisense sequences comprises proto-oncogene (ras
H, ras
K, ras
N, c-myc, bcl-2, Akt), antisense dna sequence, antisense oligonucleotide and the antisense oligodeoxyribonucleotide of somatomedin and acceptor gene thereof; Antioncogene comprises p53, Rb, PTEN, and suicide gene comprises HSV-TK (herpes simplex virus thymidine kinase) gene and CD (coli cytosine deaminase) gene; Apoptosis gene comprises p15, p16, p21
WAF-1Cytokine gene comprises GM-CSF (granulocyte macrophage colony stimulating factor) gene, TNF α (tumor necrosis factor) gene, INF (interferon) α, γ gene, IL (interleukin) 2,3,4,12,15,18 genes etc.
Target gene entraining system
Targeting non-virus carrier of the present invention is mixed with foreign DNA, just constitute the gene import system of the receptor-mediated targeting therapy of tumor of EGFR.Depend on used foreign DNA, gene import system of the present invention can be used for treating diseases such as genetic diseases or tumor, in particular for gene therapy.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical composition, and it contains one or more targeting non-virus carrier of the present invention and pharmaceutically acceptable carrier or excipient of the present invention of safe and effective amount.This class carrier comprises (but being not limited to): saline, buffer, glucose, water, glycerol, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.
As used herein, term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various excipient and diluent.This term refers to some medicament carriers like this: they itself are not necessary active component, and do not have undue toxicity after using.Suitable carriers is well known to those of ordinary skill in the art.(Mack Pub.Co. can find discussing fully about pharmaceutically acceptable excipient in N.J.1991) at Remington ' s Pharmaceutical Sciences.In addition, also may there be complementary material in these carriers, as wetting agent or emulsifying agent, pH buffer substance etc.
Pharmaceutical composition of the present invention can be made into the injection form, for example is prepared by conventional method with normal saline or the aqueous solution that contains glucose and other adjuvant.
When using, also contain one or more above-mentioned foreign DNAs in the pharmaceutical composition.
The pharmaceutical composition for preparing can carry out administration by conventional route, comprising (but being not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, Intradermal or topical.
When making pharmaceutical composition, be that target gene entraining system (effective ingredient is targeting non-virus carrier of the present invention and foreign DNA) with safe and effective amount is applied to mammal, wherein this safe and effective amount is usually at least about 1 microgram/kg body weight, and in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are within the skilled practitioners skill.
Major advantage of the present invention is:
(1) the present invention combines EGFR with the binding site of EGFR with the natural EGFR part of micromolecule polypeptide anthropomorphic dummy from the interactional mechanism of protein molecular.Compare with monoclonal antibody, the micromolecule polypeptide structure is clear, with EGFR binding site limitation, is difficult for the space steric effect between generation polypeptide and the polypeptide, does not also have the interference of Fc section.
(2) micromolecule polypeptide does not almost have antigenicity, and side effect is little, easily preparation and large-scale production and characteristics such as easy to use.
(3) the relevant polypeptide of the present invention is owing to can combine with cell surface EGFR receptor protein and may reach the function that regulate tumor cell is grown by activating or block the EGFR signal transduction pathway, thereby can be used as precursor or the pharmaceutical composition that clinical medicine is developed, it comprises code book and invents the DNA of relevant polypeptide and the antibody of the relevant polypeptide of the present invention.
(4) the relevant polypeptide of the present invention can be used as the targeting peptide and is used for gene transfer system, carries out the target gene therapy at the tumor cell of EGFR receptor high expressed.
(5) the relevant polypeptide of the present invention can be used as the targeting peptide and connects the targeted therapy effect that radionuclide, chemicals, biotoxin are used for malignant tumor and EGFR systemic-function dysfunctional disease.
(6) DNA sequence of the relevant polypeptide of the present invention and this polypeptide of encoding thereof also can be used as and detects or diagnostic reagent.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
In the present embodiment, prepare GE9, GE10 and GE11 with artificial synthesis.
With polypeptide GE9[SEQ ID NO:1] be example, its synthetic solid-phase synthesis that adopts carries out on Peptide synthesizer (Applied biosystems Inc.).
1). the polypeptide solid phase synthesis
Select resin: Fmoc-Lys (Boc) Wang resin, consumption is: 0.46mmol/g
A. deprotection base (Fmoc): the DMF solution with 20% piperidines takes off (5 ' and 15 ') twice; With DMF washing 3 times, MeOH washs 3 times resin respectively, DCM washing 3 times.
B. connect Fmoc-aminoacid
Fmoc-AA/Bop/HoBt/DIEA=1: 1: 1: 2 (corresponding to excessive 5 times of the molal quantity of resin)
With DMF washing 3 times, MeOH washs 3 times respectively for condensation 1 hour, resin, DCM washing 3 times.
The c.Kaiser test
Be then reclosing of blueness as resin, till colourless.Repeat above a, b, c step, according to polypeptide GE9[SEQ ID NO:1] aminoacid sequence, successively each aminoacid is connected.
2). cutting resin
Use TFA: H2O: phenol: EDT=92.5: 2.5: 2.5: 2.5,10ml/g cut the resin room temperature reaction 2 hours.Filter, filtrate concentrating removed TFA, the precipitation that adds diethyl ether, and the ether washing gets crude product.
3). oxidation
Make the aqueous solution of 1mmol/L, use NH
4Ac transfers pH to 7-8, and air oxidation is spent the night, and follows the tracks of through HPLC.
4). purification
Concentrate and to desalt, through the HPLC purification, concentrate, lyophilizing, dry product.
5). mass spectral analysis
Carry out mass spectral analysis, with ESI bunch of determining molecular weight of electron spray ionisation, the molecular weight size of gained polypeptide is close with theoretical value, is 1504.6, shows this polypeptide GE9[SEQ ID NO:1 just] (c-terminus has joint sequence GGGGSY).Experimental result is seen Fig. 1.
With the GE10 (c-terminus has joint sequence GGGGSY) of same procedure system, the molecular weight of mensuration is 1985.5, and is close with theoretical value, shows this polypeptide GE10[SEQ ID NO:2 just].
With the GE11 of same procedure system, the molecular weight of mensuration is 1541.2, and is close with theoretical value, shows this polypeptide GE11[SEQ ID NO:3 just].
Embodiment 2 GE9 combine test with SMMC7721 cell surface EGFR receptor-specific
With SMMC7721 cell inoculation commonly used in 96 orifice plates, 5 * 10
3Cells/well, overnight incubation is treated cell attachment, wash 3 times with PBST (PBS+0.1%Tween20), the competition group adds usefulness binding buffer liquid (the PBS+20mM HEPES+2.5mg/ml BSA of pre-cooling, pH7.4) the every hole 20 μ l of Xi Shi GE9 or EGF (containing GE9 or EGF 20pmol), non-competing group adds 20 μ l binding buffer liquid, every group of parallel three holes; Iodine mark GE9 is diluted to 1 milliliter with binding buffer liquid simultaneously, and every hole adds 20 μ l and (contains
125I GE9 0.02pmol).4 ℃ in conjunction with 3 hours.PBST washes 3 times, adds the pancreatin of 100 μ l 0.25%, digests 10 minutes, transfers to and measures in the pipe, washes with 100 μ l PBST, equally washing liquid is added in the corresponding mensuration pipe gamma counting.
Experimental result is seen Fig. 2.Show that GE9 can combine with SMMC7721 cell surface EGFR receptor generation specificity.
Embodiment 3
125I labelling GE9 combines with the EGFR protein-specific
Every hole 0.1M NaHCO
3(PH 8.6) 50 μ l wrap by 1 μ gEGFR albumen (SIGMA company) in 96 orifice plates, 4 ℃ are spent the night, PBST washes 3 times, the competition group adds the GE9 every hole 20 μ l (containing GE9 20pmol) with binding buffer liquid (PBS+20mMHEPES+2.5mg/ml BSA pH7.4) dilution of pre-cooling, non-competing group adds 20 μ l buffer, every group of parallel three holes; The while handle
125I GE9 is diluted to 1 milliliter with binding buffer liquid, and every hole adds 20 μ l and (contains
125I GE9 0.02pmol).Under the room temperature in conjunction with 1 hour.PBST washes 3 times, adds 100 μ l Glycine-HCl (PH2.2) eluting 15 minutes, transfers to and measures in the pipe, and reuse 100 μ l Glycine-HCl wash, and equally washing liquid is added in the corresponding mensuration pipe gamma counting.
The results are shown in Figure 3.Show that GE9 can combine with EGFR albumen generation specificity.
Embodiment 4 GE9 and EGFR receptor affinity detect
Inoculation SMMC7721 cell is in 96 orifice plates, 5 * 10
3Cells/well, overnight incubation is treated cell attachment, washes 3 times with PBST (PBS+0.1%Tween20), with sealing buffer (Blocking Buffer) (0.05mM PB+5mg/ml BSA) 100 μ l, 4 ℃ were sealed 1 hour, and PBST washes 3 times, added corresponding according to the every hole of following Concentraton gradient
125The GE9 of I labelling, each concentration group is established three parallel holes, and every hole reaction system is 100 μ l.4 ℃ in conjunction with 3 hours, and PBST washes 3 times, adds the pancreatin of 100 μ l 0.25%, digests 10 minutes, transfers to and measures in the pipe, washes with 100 μ l PBST, equally washing liquid is added in the corresponding mensuration pipe, and gamma is counted.According to experimental result, the dissociation constant Kd[Concentraton gradient that application Scatchard plot calculates reaction GE9 and EGFR receptor affinity size is: 0.2nM, 0.39nM, 0.78nM, 1.56nM, 3.13nM, 6.25nM, 12.5nM, 25nM, 50nM].
The results are shown in Figure 4.The GE9 that measures and the Kd of EGFR receptors bind are 1.33 * 10
-8M is far above present existing EGFR part oligopeptide.
Embodiment 5 phage clones combine with cell surface EGFR receptor-specific
Take the logarithm the SMMC7721 cell inoculation of trophophase to 96 orifice plates, 1 * 10
4Cells/well, overnight incubation is treated cell attachment, PBST washes 3 times, 37 ℃ of 10% formaldehyde fixed 30 minutes, PBST washes 3 times, and 5 minutes/time, added 37 ℃ of 0.03% hydrogen peroxide (methanol preparation) 30 minutes, PBST washes 3 times, adds 10
11Pfu shows 37 ℃ of warm baths 1 hour of phage (containing 0.I%BSA) of GE9 and GE10.PBST washes 6 times, adds HRP-mouse-anti M13 polyclonal antibody 100 μ l/ holes, 37 ℃ 1 hour.After the PBST rinsing, add the colour developing of DAB working solution lucifuge, cessation reaction when suitable of waiting to develop the color.
The results are shown in accompanying drawing 5, show that the phage of showing GE9 and G10 combines with the SMMC7721 cell of expressing EGFR.
Embodiment 6
125I labelling GE11 combines with the EGFR protein-specific
With embodiment 3 same procedure, measure
125I labelling GE11 combines with the EGFR protein-specific.
The results are shown in Figure 6, show that GE11 can combine with EGFR albumen generation specificity.
1) the business-like M13 host bacterium escherichia coli ER2738 of picking routine is inoculated in the 5ml centrifuge tube that contains 2ml LB training base.300rpm/min is to exponential phase.
2) (be dissolved in 50ul 0.1MNaHCO3, pH8.6) with 96 orifice plates, 4 ℃ are spent the night bag by EGFR and each 1ug of BSA (bovine serum albumin).
3) outwell coating buffer, (5mg/ml is dissolved in 0.1M NaHCO to add 250ul BSA
3, pH8.6), 4 ℃ 2 hours.Outwell liquid, (pH7.5 l50mMNaCl), washs 3 times for 0.5%Tween-20,50mM Tris-HCl with TBST.
4) every hole adds 1 * 10
9Phage (being dissolved in 100ul TBST), room temperature, jog 30 minutes.
5) remove liquid, with TBST washing 10 times.
6) 100ul 0.2m Glycine-HCl (pH2.2) eluting 10 minutes are used in every hole, and each adds 15ul Tris-HCl (pH9.1) neutralization.
7) phage titration: respectively get the 1ul eluent and carry out titration.10 times of gradient dilution eluents at first.Add 200ul ER2738 culture fluid in the 5ml centrifuge tube, every pipe adds the 10ul diluent, and mixing adds (every liter: 10g bacto peptone, 5g yeast extract, 5gNaCl, 1g MgCl in 45 ℃ of upper strata glue of 3ml then
26H
2O, the 7g agarose), other adds 30ul IPTG/Xgal (mixing 1.25g IPTG and 1g Xgal in the 25ml dimethylformamide).The vibration mixing.Be laid on the 90mmLB agar plate immediately.Be inverted plate in 37 ℃ of incubators, lucifuge is spent the night.Calculated clone's number (locus coeruleus number) in second day.
Fig. 7 as a result, wherein GE31 is the contrast phage.Illustrate that the phage of showing GE9, GE10 and GE11 can the combination of EGFR specificity.
The structure of embodiment 8 non-virus carriers
A. the acquisition of each component
(1) part oligopeptide GE's is synthetic:
Press the identical method of embodiment 1, adopt the Peptide synthesizer of American AB I company and synthesize according to its polypeptide synthetic operation handbook, column chromatography for separation obtains oligopeptide GE9, GE10 and GE11, and its aminoacid sequence is respectively SEQ ID NO:1,2 and 3.
(2) poly-L-lysine (Poly-L-lysine is abbreviated as P.L.)
Available from U.S. SIGMA company, molecular weight distribution is 15000-30000 dalton.
(3)HA20
Adopt the Peptide synthesizer of American AB I company and synthesize according to its polypeptide synthetic operation handbook, column chromatography for separation obtains oligopeptide HA20, and its aminoacid sequence is GLFEA IAEFI EGGWE ELIEG (SEQ IDNO:4).
B. the preparation of binary complex
At first, GE (GE9 or GE10 or GE11), HA20 and poly-L-lysine (Poly-L-lysine is abbreviated as P.L.) are dissolved among the PSB (0.1M sodium phosphate buffer, pH7.4 contains 0.1MNaCl), then, follow these steps to react.
(1)P.L.+SPDP→P.L.-PDP
Annotate: SPDP is N-butanimide-3-(2-pyridine dithio)-propionic ester.
P.L. the mol ratio with SPDP (N-) is 1: 5, and the response time is 2 hours, and temperature is 25 ℃.Reaction finishes the back by the unreacted SPDP of dialysis removal.The P.L.-PDP lyophilization.
(2) P.L.-PDP dry powder is dissolved among the PSB
P.L.-PDP+DTT→P.L.-SH
Annotate: DTT is a dithiothreitol, DTT.
DTT is excessive in the reaction, and the response time is 1 hour, and temperature is 25 ℃.Reaction finishes the back by the unreacted DTT of dialysis removal.The product lyophilization.
(3)GE+SPDP→GE-PDP
HA20+SPDP→HA20-PDP
The mol ratio of GE, HA20 and SPDP is 1: 5, and the response time is 2 hours, and temperature is 25 ℃.Unreacted SPDP is removed in bag filter dialysis by MWC01000 after reaction finishes.The product lyophilization.
(4) GE-PDP, HA20-PDP and P.L.-SH dry powder all are dissolved among the PSB.
GE-PDP+P.L.-SH→P.L.-GE
HA20-PDP+P.L.-SH→P.L.-HA20
The mol ratio of GE-PDP, HA20-PDP and P.L.-SH is 3: 1, and the response time is 24 hours, and temperature is 25 ℃.Reaction finishes the back by dialysis unreacted GE-PDP of removal and HA20-PDP.
C. the preparation of targeting non-virus carrier
Mix P.L.-GE and P.L.-HA20 in 1: 1,0.8: 1.2,1.2: 0.8 with mass ratio respectively, promptly get required non-virus carrier.
Perhaps, GE, polylysine and HA20 are mixed, form required non-virus carrier by mass ratio 1: 1.5: 1.
The extracting and the purification of embodiment 9 plasmid DNA
The isolation and purification of used recombinant eukaryon expression vector plasmid DNA all utilizes the Maxi plasmid kit of QIAGEN company and the pure product of DNA that obtain in the present embodiment.
Select monoclonal from the LB flat board and be inoculated in the 5ml LB culture medium that contains ampicillin, 37 ℃, the 250rpm jolting is spent the night.Transfer at 1: 1000 in the 200ml LB culture medium that contains ampicillin, 37 ℃, 250rpm jolting 16 hours.Shift bacterium liquid in the 500ml centrifuge tube, 4,000rpm, 4 ℃ centrifugal 10 minutes, abandon supernatant, collect thalline.Precipitation is resuspended in 10ml buffer P1 (50mM TrisCl, pH8.0,10mM EDTA pH8.0,100 μ g/ml Rnase A).(200mM NaOH puts upside down 4-6 time after 1%SDS) gently, and mixing left standstill under the room temperature 5 minutes to add 10ml buffer P2.Add 10ml buffer P3 (3.0M NaAc, pH5.5), put upside down mixing after, be transferred to the 50ml centrifuge tube, 10,000rpm, 4 ℃ centrifugal 30 minutes, get supernatant.Supernatant is aseptically filled in the QIAGEN Maxi plasmid extraction post through four layers and (uses 10ml buffer QBT balance in advance), allow under its spontaneous current, enter in the post fully Deng supernatant, again with buffer QC 30ml washing secondary, add buffer QF15ml at last, collect effluent, add 0.7 times of volume isopropyl alcohol, 10,000rpm, 4 ℃ centrifugal 30 minutes, abandon supernatant, 75% washing with alcohol precipitation 1 time, natural drying under the room temperature is dissolved among the TE (pH8.0) of an amount of volume the quality of the firm DNA of 1% agarose gel electrophoresis, its purity of UV spectrophotometer measuring is also quantitative ,-20 ℃ of preservations.
The structure of embodiment 10 quaternary complex gene import systems
1) after .GE-P.L. and HA20-P.L. were dissolved in water, through 0.22 μ m filter filtration sterilization ,-20 ℃ stored for future use.All plasmids extract and purification through the QIAGEN post, the ethanol of 2 times of volumes of reuse and the 3M NaAc of 1/3 volume precipitation, and after 75% ethanol is washed once, air drying.With an amount of MilliQ water dissolution, make concentration become 0.2 μ g/ μ l.
2). the optimum quality ratio when non-virus carrier and DNA form complex between the two.
0.2 μ g CMV-β-gal plasmid DNA was mixed with mass ratio with non-virus carrier in 1: 0.5,1: 1,1: 1.5,1: 2,25 ℃ left standstill 30 minutes, with the retardance situation of 1% agarose gel electrophoresis identification of dna, determined optimum quality ratio.
3). prepare quaternary complex gene transfer system by above-mentioned definite proportioning.
Embodiment 11 GE gene import systems transduction CMV-β-gal gene in vitro imports experiment
1). cell culture
The SMMC7721 passage is cultivated: the SMMC7721 cell with 0.25% trypsinization after, be inoculated in the Tissue Culture Flask, adding contains the DMEM complete culture solution of 10% calf serum, at 37 ℃, 5%CO
2Cultivate in the incubator.
2). external importing experiment
Cultured cell with 0.25% trypsinization after, be inoculated in six orifice plates every hole 1.5 * 10
5, the cell full scale was about 60% o'clock in second day, added the quaternary complex gene transfer system be equivalent to 2 μ g DNA (promptly respectively based on three kinds of quaternary complex gene transfer systems of GE9, GE10, GE11) in the 1ml culture fluid.After the transfection 24 hours, renew the bright DMEM complete culture solution that contains 10% calf serum, continue to cultivate 24 hours.Cell is washed 2 times with PBS then, add freshly prepared fixative and fix 10 minutes in room temperature, reuse PBS rinsing 3 times, each 15 minutes, with thorough removal fixative, blot PBS on tumor piece and the internal organs with filter paper, add freshly prepared dyeing liquor in 37 ℃ of insulations after 24 hours, the situation that the observation exogenous gene imports (form: 1mg/ml X-gal, 5mM K by dyeing liquor
4[Fe (CN)
6], 5mMK
3[Fe (CN)
6], 2mM MgCl
2).
The result shows, the cell of transduction β-gal gene of the present invention is blue, the matched group that adds normal saline and the simple plasmid respectively importing of source gene of then not regarding sb. as an outsider.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition 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 institute restricted portion equally.
Sequence table
＜110〉Shanghai Xinshijie Gene Techn Development Co., Ltd.
＜120〉with the bonded part oligopeptide of human epidermal growth factor acceptor EGFR specificity
<130>037800
<160>7
<170>PatentIn?version?3.1
<210>1
<211>9
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
＜223〉be incorporated into the part oligopeptide GE9 of EGFR receptor
<400>1
Cys?Lys?Ser?Pro?Glu?Pro?Gln?His?Cys
1 5
<210>2
<211>12
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉be incorporated into the part oligopeptide GE10 of EGFR receptor
<400>2
Leu?His?Leu?Trp?Val?Pro?Glu?Pro?Trp?Thr?Gln?Thr
1 5 10
<210>3
<211>12
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉be incorporated into the part oligopeptide GE11 of EGFR receptor
<400>3
Tyr?His?Trp?Tyr?Gly?Tyr?Thr?Pro?Gln?Asn?Val?Ile
1 5 10
<210>4
<211>20
<212>PRT
＜213〉artificial sequence
<220>
<221>MISC_FEATURE
<222>(1)..(20)
＜223〉the endocytosis corpusculum discharges oligopeptide HA20
<400>4
Gly?Leu?Phe?Glu?Ala?Ile?Ala?Glu?Phe?Ile?Glu?Gly?Gly?Trp?Glu?Glu
1 5 10 15
Leu?Ile?Glu?Gly
20
<210>5
<211>27
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉coded sequence of part oligopeptide GE9
<400>5
tgcaaaagcc?cggaaccgca?acattgc 27
<210>6
<211>36
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉coded sequence of part oligopeptide GE10
<400>6
ctgcatctgt?gggttccgga?accgtggacc?cagacc 36
<210>7
<211>36
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉coded sequence of part oligopeptide GE11
<400>7
aatcacattc?taaggcgtat?acccatacca?atgata 36
Claims (7)
1. product, it is the gene transfer system of the targeting of EGF-R ELISA EGFR mediation, it is characterized in that it comprises the part oligopeptide that (a) specificity is incorporated into the EGFR receptor, wherein said part oligopeptide is the part oligopeptide of aminoacid sequence shown in SEQ ID NO:1; (b) polycation polypeptide or albumen; (c) the endocytosis corpusculum discharges oligopeptide; (d) foreign DNA.
2. product as claimed in claim 1, it is characterized in that, the polycation polypeptide is selected from down group: poly-D-lysine, poly-acetimide, protamine, histone, adenovirus pV albumen, adenovirus pVII albumen, m μ albumen, and through polyethyleneglycol modified above-mentioned albumen and composition thereof.
3. product as claimed in claim 1 is characterized in that, described foreign DNA is selected from down group: the recombinant eukaryotic expression plasmid DNA of antioncogene, apoptosis gene, cytokine gene, oncogene antisense sequences, and composition thereof.
4. product as claimed in claim 1 is characterized in that, described foreign DNA is selected from down group:
(i) antioncogene, described antioncogene is selected from p53 or Rb;
(ii) apoptosis gene, described apoptosis gene is selected from Caspase, p15, p16 or p21
WAF-1
(iii) cytokine gene, described cytokine gene gene is selected from GM-CSF gene, TNF α gene, INF α gene, INF γ gene, IL2, IL3, IL4, IL12, IL15 or IL18 gene;
The (iv) antisense sequences of oncogene, the antisense sequences of described oncogene is selected from the antisense sequences of oncogene ras, c-myc or Akt; And/or
(v) recombinant eukaryotic expression plasmid, described recombinant eukaryotic expression plasmid are selected from viral recombinant eukaryotic expression plasmid and are the non-viral recombinant eukaryotic expression plasmid of cis-regulating element with SV40 or CMV promoter.
5. product as claimed in claim 1 is characterized in that, described component (a) part oligopeptide, (b) polycation polypeptide and (c) both or the three that discharge in the oligopeptide of endocytosis corpusculum exist with the fusion rotein form.
6. product as claimed in claim 1 is characterized in that, described endocytosis corpusculum discharges oligopeptide and is selected from down group: HA20 or VP-1.
7. an oligopeptide is characterized in that, it is incorporated into the EGFR receptor, and its aminoacid sequence is shown in SEQID NO:1.
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| CN102443056B (en) | 2010-10-15 | 2015-04-29 | 上海市肿瘤研究所 | Exon deleted variant of epidermal growth factor receptor |
| CN103333895B (en) * | 2013-01-23 | 2015-07-01 | 南方医科大学 | Nucleic acid aptamer specifically binding to human epidermal growth factor acceptor III type mutant and applications thereof |
| CN109265555B (en) * | 2018-09-04 | 2020-10-27 | 武汉原生药谷生物医药科技有限公司 | Construction and application of enhanced small molecule antibody |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1181422A (en) * | 1996-10-31 | 1998-05-13 | 上海市肿瘤研究所 | Gene transfer carrier structured by polypeptide in conjunction with growth factor receptor |
-
2004
- 2004-03-31 CN CNB2004100173346A patent/CN100356981C/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1181422A (en) * | 1996-10-31 | 1998-05-13 | 上海市肿瘤研究所 | Gene transfer carrier structured by polypeptide in conjunction with growth factor receptor |
Non-Patent Citations (1)
| Title |
|---|
| 表皮生长因子受体介导的新型肝癌靶向性基因转移 马春红，孙汶生等.基础医学与临床，第24卷第1期 2004 * |
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| CN1676166A (en) | 2005-10-05 |
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