WO2016011935A1 - 人fgfr2c胞外段蛋白质及其编码基因与应用 - Google Patents

人fgfr2c胞外段蛋白质及其编码基因与应用 Download PDF

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WO2016011935A1
WO2016011935A1 PCT/CN2015/084692 CN2015084692W WO2016011935A1 WO 2016011935 A1 WO2016011935 A1 WO 2016011935A1 CN 2015084692 W CN2015084692 W CN 2015084692W WO 2016011935 A1 WO2016011935 A1 WO 2016011935A1
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fgfr2c
amino acid
acid sequence
protein
vector
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PCT/CN2015/084692
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汪炬
陈安安
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暨南大学
广州圣露生物技术有限公司
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Priority to US15/327,881 priority patent/US20170204157A1/en
Publication of WO2016011935A1 publication Critical patent/WO2016011935A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of genetic engineering, and in particular to the extracellular domain of human FGFR2c 146-356 and its coding gene and application.
  • EGFR is an expression product of the proto-oncogene c-erbB1 and is a member of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4).
  • the HER family plays an important regulatory role in the process of cell physiology.
  • the EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation and differentiation. Loss of protein tyrosine kinase function such as EGFR or abnormal activity or cell localization of key factors in related signaling pathways can cause malignant tumors, diabetes, immunodeficiency and cardiovascular diseases. EGFR is also an important molecular target in the treatment of malignant tumors.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems in the prior art, and as a result, have found that proteins and variants thereof comprising amino acid sequences 146-356 of the extracellular domain of human FGFR2c are capable of inhibiting EGF signaling but not activating FGF signaling. Thus, the present invention has been completed.
  • the present invention includes:
  • a vector comprising the nucleic acid of item 4.
  • a host cell comprising the vector of item 5.
  • the host cell according to Item 6 characterized in that the host cell is any one of CHO cells, Escherichia coli cells, insect cells, and yeast cells.
  • a fusion protein which is a fusion protein of the isolated protein according to any one of items 1 to 3 with other polypeptides.
  • the fusion protein according to any one of items 1 to 3, which is a fusion protein of a human immunoglobulin epitope tag sequence or a human immunoglobulin Fc segment.
  • the isolated protein according to any one of items 1 to 3, the nucleic acid according to item 4, the vector of item 5, the host cell of item 6 or 7, or the fusion protein of item 8 or Use in the preparation of a medicament for the treatment of a malignant tumor.
  • a pharmaceutical composition for treating a malignant tumor which comprises the isolated protein according to any one of items 1 to 3, the nucleic acid according to item 4, the carrier according to item 5, or the item 6 or The host cell or the fusion protein of item 8 or 9 as an active ingredient, and a pharmaceutically acceptable carrier.
  • Figure 1 is a SDS-PAGE diagram of the extracellular domain of human FGFR2c 146-356 (sFGFR2c) in Example 1; wherein lane M is the protein Marker, lane 1 is the induced wild type (wsFGFR2c), and lane 2 is the uninduced wild Type, lane 3 is the induced S252W mutant (msFGFR2c) and lane 4 is the uninduced S252W mutant.
  • sFGFR2c the protein Marker
  • lane 1 is the induced wild type (wsFGFR2c)
  • lane 2 is the uninduced wild Type
  • lane 3 is the induced S252W mutant (msFGFR2c)
  • lane 4 is the uninduced S252W mutant.
  • Figure 2 is a western blot hybridization diagram of the protein of sFGFR2c in Example 1; wherein Lane 1 is wild type and Lane 2 is S252W mutant.
  • Figure 3 is a comparison diagram of the stability comparison of sFGFR2c (146-356), sFGFR2c (147-366aa) and sFGFR2c (151-377aa) and tumor cell inhibition effect in Example 7; wherein Figure A is an electropherogram of the stability of sFGFR2c, Figure B is An electropherogram of the stability of sFGFR2c (147-366aa), and Figure C is an electropherogram of the stability of sFGFR2c (151-377aa).
  • Figure 4 is a co-IP map of the binding ability of sFGFR2c to EGFR in Example 8; wherein wsFGFR2c is wild-type human FGFR2c extracellular domain 146-356 and msFGFR2c is S252W mutant FGFR2c extracellular segment 146-356.
  • Figure 5 is a graph showing the effect of sFGFR2c on the proliferation of DU145 cells in Example 8; wherein, wsFGFR2c is wild-type human FGFR2c extracellular domain 146-356, and msFGFR2c is S252W mutant human FGFR2c extracellular domain 146-356, ⁇ represents P ⁇ 0.01 compared with the blank control group; ⁇ represents P ⁇ 0.01 compared with the EGF alone induction group.
  • Figure 6 is a diagram showing the hybridization of the effect of human FGFR2c extracellular domain 146-356 on the EGFR/ERK signaling pathway in Example 8; wherein wsFGFR2c is wild-type human FGFR2c extracellular domain 146-356, and msFGFR2c is S252W mutant human FGFR2c Extracellular segment 146-356.
  • Figure 7 is a graph showing the results of inhibition of FGFRs and ERK phosphorylation by sFGFR2c by western blot.
  • Figure 8 is a graph showing the results of an experiment for detecting the interaction of the extracellular domain 146-356 of FGFR2c with EGFR by isothermal titration calorimetry (iTC).
  • the invention provides an isolated protein comprising the amino acid sequence:
  • the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 is amino acid 146-356 (wsFGFR2c) of the extracellular domain of human FGFR2c
  • the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 is in SEQ ID Among the proteins having the amino acid sequence of NO: 1, a protein (msFGFR2c) in which the serine (S) at position 252 of the full-length segment of the wild type human FGFR2c is mutated to tryptophan (W).
  • proteins consisting of the amino acid sequence of SEQ ID NO: 1 or 2 are collectively referred to as human FGFR2c extracellular domain 146-356.
  • Fibroblast growth factor (FGF receptor) (FGFR) is a receptor on the membrane, and its extracellular portion can bind to a specific ligand, while the intracellular portion has tyrosine kinase activity. Binding of the extracellular portion to the ligand activates the dimerization and phosphorylation of the receptor and leads to activation of downstream signals, thereby regulating the expression of the target gene.
  • the extracellular domain of FGFR2c is the extracellular portion of the FGF receptor 2c subtype, which binds to the ligand and reduces the effective concentration of the ligand, thereby inhibiting FGF signaling.
  • the inventors have demonstrated that the isolated proteins described above are capable of inhibiting EGF signaling, but not activating, preferably inhibiting, FGF signaling.
  • the invention provides an isolated protein comprising the amino acid sequence described below and having the function of inhibiting EGF signaling but not activating FGF signaling:
  • the amino acid sequence of SEQ ID NO: 1 or 2 has 80% or more, preferably 85% or more, more preferably 87.8%. More preferably, the amino acid homology of 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, still more preferably 98% or more, still more preferably 99% or more, still more preferably 99.5% or more Sequence; or
  • the above-described isolated protein is referred to as a variant of the human FGFR2c extracellular segment 146-356.
  • the variant of human FGFR2c extracellular segment 146-356 is derived from a human.
  • the amino acid substitutions may be conservative substitutions, i.e., replacement of a particular amino acid residue with a residue having similar physicochemical characteristics.
  • conservative substitutions include substitutions between amino acid residues containing aliphatic groups (eg, substitutions between Ile, Val, Leu, or Ala), substitutions between polar residues (eg, Lys and Arg, Glu) And mutual substitution between Asp, Gln and Asn).
  • variants in which amino acid deletions, substitutions, insertions, and/or additions can be carried out by, for example, well-known site-directed mutagenesis of DNA encoding wild type proteins (see, for example, Nucleic Acid Research, Vol. 10, No. 20, p. 6487- 6500, 1982, which is incorporated by reference in its entirety to the present specification, or by the method of artificially synthesizing a protein.
  • one or more amino acids refers to an amino acid which can be deleted, substituted, inserted and/or added by site-directed mutagenesis or artificial synthesis, for example, 1 to 20 amino acids, preferably 1 to 15 amino acids, It is more preferably 1 to 10 amino acids, still more preferably 1 to 8 amino acids, still more preferably 1 to 2 amino acids, still more preferably 1 amino acid.
  • the % homology of the two amino acid sequences can be determined by visual and mathematical calculations. Or the percent homology of the two polypeptide sequences can be based on the algorithm of Needleman, SB and Wunsch, CD (J. Mol. Bol., 48: 443-453, 1970), available from the University of Wisconsin Genetics
  • the GAP computer program obtained by the computer group (UWGCG) is determined by comparing the sequence information.
  • Preferred default parameters for the GAP program include: (1) Henikoff, S. and Henikoff, JG (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992) scores/matrices, blosum62; (2) 12 points for one vacancy; (3) 4 points for consecutive vacancies; and (4) no decency for end vacancies.
  • sequence information can be compared and determined using the BLAST program described in Altschul et al. (Nucl. Acids. Res., 25, p. 3389-3402, 1997).
  • the program can be used on the website at the Nantional Center for Biotechnology Information (NCBI) or DNA Data Bank of Japan (DDBJ) website.
  • NCBI Nantional Center for Biotechnology Information
  • DDBJ DNA Data Bank of Japan
  • various conditions (parameters) for performing homology search using the BLAST program are described in detail, and some settings can be appropriately changed, but the search is usually performed by default values.
  • the % homology of the two amino acid sequences can also be determined by a program such as genetic information processing software GENETYX Ver. 7 (manufactured by GENETYX) or a FASTA algorithm. At this time, it can also be retrieved with default values.
  • stringent conditions means a strip which forms a so-called specific hybrid and does not form a non-specific hybrid. Pieces.
  • Examples of stringent conditions include conditions under which highly homologous DNA hybridizes to each other, for example, not less than 80% homologous, preferably not less than 90% homologous, more preferably not less than 95% DNA homologous, still more preferably not less than 97% homologous, particularly preferably not less than 99% homologous, hybridizes to each other, and DNA having lower homology than the above does not hybridize with each other, or washing conditions of typical Southern hybridization That is, at a salt concentration and temperature corresponding to 1xSSC, 0.1% SDS at 60 ° C, preferably 0.1 x SSC, 0.1% SDS at 60 ° C, more preferably 0.1 x SSC, 0.1% SDS at 68 ° C, preferably 2 or 3 conditions.
  • Whether or not the function of "inhibiting the EGF signal but not activating (preferably suppressing) the FGF signal” can be determined by, for example, the method described in the examples.
  • the invention provides an isolated nucleic acid encoding the above-described human FGFR2c extracellular domain 146-356 or a variant thereof.
  • the isolated nucleic acid may be single-stranded or double-stranded; it may be DNA, RNA, or a hybrid of DNA and RNA.
  • the isolated nucleic acid can be prepared by a synthetic method, or can be produced, for example, by a genetic engineering method.
  • amino acid sequence described in SEQ ID NO: 1 or 2 above can be encoded by the base sequence set forth in SEQ ID NO: 3 or 4, respectively.
  • the invention provides a vector comprising the nucleic acid.
  • the carrier includes a plasmid, a phage, an animal virus, and the like.
  • the vector is an expression vector.
  • the expression vector includes a prokaryotic expression vector and a eukaryotic expression vector, preferably a pET3c vector, a pCDNA3.1 vector, a pIRESneo3 vector, a pPICZ ⁇ A vector or a pFastBac vector.
  • the invention provides a host cell comprising the vector.
  • host cell There is no particular limitation on the type of host cell, and those which are conventionally used by those skilled in the art.
  • Examples of the host cell include CHO cells, Escherichia coli cells, insect cells, and yeast cells.
  • the invention provides fusion of the above-described human FGFR2c extracellular domain 146-356 or a variant thereof with other polypeptides Protein.
  • the additional polypeptide is a human immunoglobulin epitope tag sequence or a human immunoglobulin Fc segment.
  • the fusion protein can be prepared by conventional methods in the art.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above human FGFR2c extracellular domain 146-356 or a variant thereof, the above nucleic acid, the above vector, the above host cell or the above fusion protein as an active ingredient
  • a pharmaceutically acceptable carrier is included in the present invention.
  • the pharmaceutical composition of the invention can be used to treat malignant tumors. Accordingly, in a further aspect, the present invention provides the above human FGFR2c extracellular domain 146-356 or a variant thereof, the above nucleic acid, the above vector, the above host cell or the above fusion protein in the preparation of a medicament for the treatment of a malignant tumor use.
  • malignant tumors include prostate cancer, oral cancer, nasal mucosa cancer, tracheal cancer, bronchial cancer, lung cancer, esophageal cancer, gastric cancer, colon cancer, small intestine cancer, liver cancer, cholangiocarcinoma, gallbladder cancer, pancreatic cancer, and renal cancer.
  • Specific examples of the pharmaceutically acceptable carrier include sterile water, physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavor enhancer, excipient, carrier, preservative, and binding agent. Wait.
  • This example describes the preparation of wild-type wsFGFR2c and S252W mutant msFGFR2c.
  • RNA of wild-type FGFR2c extracellular domain was extracted from human placenta tissue (from a hospital in Guangdong by a maternal consent to get fresh placenta tissue) by Trizol method, and a cDNA library was established.
  • RNA pre-denaturation On the PCR instrument under the condition of 65 ° C for 10 min. Insert into the ice immediately after pre-denaturation. Then, other components in the reverse transcription reaction are added, and a reverse transcription reaction is carried out to obtain cDNA. details as follows:
  • Reverse transcription PCR program 30 ° C, 10 min; 42 ° C, 1 h; 70 ° C, 10 min.
  • the primer synthesis in the present invention is all from Beijing Liuhe Huada Gene Technology Co., Ltd.
  • restriction enzymes in the present invention are all derived from TaKaRa.
  • the wild type wsFGFR2c gene primer sequence is as follows:
  • F1 5'-CG CATATG AACAAGAGAGCACCATAC-3'; the horizontal line is the Nde I restriction site;
  • R1 5'-AT GGATCC CTATTA CAGAACTGTCAACCATGC -3 '; the underlined BamH I restriction site is.
  • R2 5'-ATGGGCCGGTGAGGCCATCGCTCCACAA-3'.
  • the cDNA obtained in step 1 was subjected to PCR using wild-type wsFGFR2c primers (F1 and R1) to obtain a wild-type wsFGFR2c gene.
  • the S252W mutant msFGFR2c gene was amplified by the overlap-type extension PCR method using the wild type wsFGFR2c gene as a template and the mutant PCR primers, respectively.
  • the first step of the PCR reaction system is the first step of the PCR reaction system:
  • reaction conditions were: 96 ° C for 5 min; 94 ° C for 15 s, 60 ° C for 15 s, 72 ° C for 5 s, 31 cycles; 72 ° C for 10 min.
  • the 1% concentration of agarose was electrophoresed, the gel was cut, and DNA was recovered using a DNA gel recovery kit (TIANGEN, DP209), and the recovered DNA fragment was detected by agarose gel electrophoresis and ultraviolet spectrophotometer for concentration and purity.
  • the OD 260 / OD 280 ratio should be 1.7-1.9.
  • the PCR amplified sequence was digested with NDE I and BamH I simultaneously with pET3c vector (from Invitrogen, USA), and the reaction conditions were: 37 ° C water bath for 4 h;
  • the pET3c plasmid and the FGFR2c gene were digested with Nde I and BamH I, respectively, and ligated with T4 DNA ligase.
  • the reaction system was prepared according to the T4 DNA ligase instructions, and the reaction conditions were 16 ° C water bath for 12 h to obtain a ligation product.
  • the BL21(DE3) expression strain was inoculated into a sterilized LB liquid medium containing a mass-to-volume ratio of 0.1% Amp at a ratio of 1:50 by volume, and cultured at 37 ° C, shaking at 200 rpm.
  • the expression of the target protein was identified by SDS-PAGE electrophoresis, and the results are shown in Fig. 1. The results showed that IPTG could induce BL21(DE3) expression strain to express the extracellular domain of FGFR2c 146-356.
  • the extracellular domain of FGFR2c 146-356 protein is expressed in the form of inclusion bodies, and the active form of FGFR2c extracellular domain 146-356 protein is obtained by inclusion body washing and renaturation technique.
  • the method can be referred to Patent Example ZL200710029286.6 Example 1).
  • This example describes the preparation of a latent glycosylated form of the FGFRc extracellular 146-356 polypeptide by recombinant expression of the wild-type wsFGFR2c, S252W mutant msFGFR2c gene in mammalian cells.
  • the primer design is as follows:
  • the vector pCDNA3.1(-) (purchased from Invitrogen, USA) was used as an expression vector;
  • PCR was carried out using F3 and R3 as primers, and the reaction system and conditions were the same as in Example 1.
  • the obtained wild-type wsFGFR2c gene and S252W mutant msFGFR2c gene were ligated to pCDNA3.1(-), respectively, and transformed into E. coli DH5 ⁇ competent cells.
  • the resulting vector was called pCDNA3.1-FGFR2c, pCDNA3.1-FGFR2c- S252W, (the specific procedure is the same as in Example 1, the double-cut enzymes used are BamH I and Hind III).
  • pCDNA3.1-FGFR2c and pCDNA3.1-FGFR2c-S252W were transfected into 293 cells, respectively.
  • the plasmid was extracted according to the method of removing the endotoxin plasmid miniprep kit (purchased from OMEGA) to obtain pCDNA3.1-FGFR2c, pCDNA3.1-FGFR2c-S252W plasmid;
  • 2 dilution of the plasmid to be transfected calculated according to the amount of each well of the six-well plate: 4 ⁇ g of the plasmid to be transfected was added to 250 ⁇ L of opti-MEM medium in a proportional dilution;
  • the culture supernatant was collected by centrifugation at 18000 rpm for 30 min at 4 ° C.
  • the protein purification steps were as follows:
  • heparin affinity chromatography column (GE 17-0998-01 50ml), rinse the column with double distilled water for 3 column volumes, and equilibrate the column with affinity chromatography equilibration solution at a flow rate of 5 ml/min and equilibrate at least 3 column volumes.
  • the supernatant obtained in the step (2) is loaded, and after the completion of the sample, the column volume is further washed with the affinity chromatography balance solution, and replaced with a heparin eluate for elution, and a single wash is collected at a wavelength of 280 nm.
  • De-peaking, wild-type wsFGFR2c polypeptide and S252W mutant msFGFR2c polypeptide were obtained and stored at -70 ° C for subsequent experiments.
  • the flow rate was 5 ml/min.
  • the downstream primer R4 5'-GCGC GAATTC TCATTA CAGAACTGTCAACCATGC-3' is a EcoR I restriction site.
  • the supernatant of the cell culture medium was harvested in a 1.5 L culture volume, and 500 ml was filtered through a 0.45 ⁇ m filter, and the target protein was purified by a heparin affinity chromatography column according to the method of Example 2, and identified by Western Blot.
  • the results indicate that the extracellular domain of FGFR2c 146-356 can be specifically recognized by FGFR antibodies.
  • Upstream primer F5 5'-ATAT CTCGAG GCCGCCACC ATG AACAAGAGAGCACCATAC-3'; the horizontal line is the Xho I restriction site;
  • Downstream primer R5 5'-GCGC TCTAGA TCATTA CAGAACTGTCAACCATGC-3'; the horizontal line is the Xba I restriction site.
  • the vector used was the Pichia pastoris expression vector pPICZ ⁇ A (Invitrogen).
  • the purified plasmid was linearized with Sac I enzyme digestion enzyme (10 ⁇ buffer 2 ⁇ l, plasmid 10 ⁇ l, Sac I 1 ⁇ l, plus ddH 2 0 to 20 ⁇ l), and electrotransformed into Pichia pastoris X33 competent cells, respectively.
  • the vectors pFastBac-FGFR2c and pFastBac-FGFR2c-S252W were obtained according to the method of Example 3 (primer as in Example 3, pFastBac vector from Invitrogen).
  • Escherichia coli competent DH10Bac (from Invitrogen) was taken out from -80 ° C and then dissolved on ice; 5 ⁇ L of the plasmid was added to E. coli competent DH10Bac under sterile conditions, placed on ice for 30 min, and heat shocked at 42 ° C for 45 s. Immediately after heat shock, put on ice and let stand for 2 minutes; add 0.9 ml of room temperature SOC medium (Cat. No.
  • Primer design is as follows: both 5'-3'
  • F10-FGFR2c ATAT ACTAGT ATG GATGACGACGACAAG AACAAGAGAGCACCATAC; the horizontal line is the SpeI restriction site;
  • R10-FGFR2c CGACCCACCACCGCCGGAGCCACCGCCACC CAGAACTGTCAACCATGC;
  • F11-Fc GGCGGTGGTGGGTCGGGTGGCGGCGGATCTCCCAAGAGCTGCGACAAG;
  • R11-Fc ATAT GAATTC TCATTACTTGCCGGGGGACAGG; the horizontal line is the EcoR I restriction site;
  • reaction conditions were: 96 ° C for 5 min; 94 ° C for 15 s, 60 ° C for 15 s, 72 ° C for 5 s, 31 cycles; 72 ° C for 10 min.
  • Figure 3B is a stable electrophoresis pattern of S252W mutant msFGFR2c (147-366aa) and wild-type FGFR2c (147-366aa) prepared according to the literature.
  • Lane 1 is a control S252W mutant type msFGFR2c (147-366), lane 2 3, 4, and 5 are S252W mutant msFGFR2c (147-366) samples 1, 3, 5, and 7 respectively;
  • Lane 6 is wild type FGFR2c (147-366) as control, lanes 7, 8, 9, and 10 They were samples of the first, third, fifth, and seventh days of wild-type FGFR2c (147-366).
  • Figure 3C is a stable electrophoresis pattern of S252W mutant msFGFR2c (151-377aa) and wild-type FGFR2c (151-377aa) prepared according to the literature.
  • Lane 1 is a control S252W mutant type msFGFR2c (151-377aa)
  • lane 2 3, 4, and 5 are S252W mutant msFGFR2c (151-377aa) samples 1, 3, 5, and 7 respectively
  • Lane 6 is wild type FGFR2c (151-377aa) as control, lanes 7, 8, 9, and 10 They were samples of the first, third, fifth, and seventh days of wild-type FGFR2c (151-377 aa), respectively.
  • the extracellular domain of FGFR2c 146-356 constructed by the present invention is more stable than the extracellular segment of FGFR2c (147-366aa) and the extracellular segment of FGFR2c (151-377aa), which is beneficial to improve the yield of the process.
  • the magnetic beads were washed with PBS containing 0.05% (v/v) Tween, and the solution was changed for 5 minutes, and repeated 8 times. After that, it was washed twice with PBS for 5 min each time.
  • Reading plate first prepare CCK8 working solution according to CCK8 instruction, then absorb the solution of medium per well, add CCK8 working solution 110 ⁇ L/well, incubate in incubator at 37 °C for 4h, remove gently shake, in enzyme Read 450/570nm dual-wavelength absorbance in the standard instrument.
  • Co-IP detects the binding of EGFR to sFGFR2c (see Figure 4).
  • Co-IP assay was used to detect the binding ability of endogenously expressed EGFR to sFGFR2c in DU145 cells. As a result, it was found that in the presence of EGF, the binding ability of msFGFR2c (i.e., S252W mutant type msFGFR2c, the same below) to EGFR was weakened (see Fig. 3B), and the same was true for wsFGFR2c (i.e., wild type wsFGFR2c).
  • msFGFR2c i.e., S252W mutant type msFGFR2c, the same below
  • EGF induced proliferation of DU145 cells and when sFGFR2c was added for induction, it inhibited the proliferation of DU145 (see Figure 5).
  • concentration of EGF was 5 ng/mL
  • the inhibition effect on EGF-induced proliferation was more obvious with the increase of sFGFR2c concentration, especially msFGFR2c.
  • concentration reached 160 ng/mL the inhibition was the best, and the inhibition rate was compared with the control group. Up to 21.1%, when the concentration induced by msFGFR2c continued to increase, the inhibitory effect decreased.
  • the inhibitory effect of wild-type wsFGFR2c was relatively weak.
  • the relative growth rate was 102.5% at 320 ng/mL compared with the control group.
  • the inhibitory effect was also attenuated.
  • iTC isothermal titration buffer
  • the dialysis completed protein was centrifuged at 18,000 rpm for 30 min at 4 ° C for ITC experiments.
  • 200 ul of EGFR was added to the sample cell at a sample concentration of 20 ⁇ mol/L.
  • 150 ⁇ mol/L of msFGFR2c 40 ⁇ L was added to the loading needle for titration.
  • the reaction conditions were titration of 0.4 ⁇ L for the first time, and the other titration was 3 ⁇ L each time, and titration was performed every 2 minutes for a total of 14 times.

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Abstract

本发明提供了一种分离的FGFR2c胞外段蛋白质，该胞外段蛋白质包含野生型的或发生了S252W突变的人FGFR2c胞外段第146-356位氨基酸。本发明还提供了编码该胞外段白质的核酸、载体和宿主细胞，以及相应的***的药物组合物。

Description

人FGFR2c胞外段146-356及其编码基因与应用
本专利申请要求中国专利申请CN201410347938.0(发明名称：FGFR2c胞外段类似物及其编码基因与应用，申请日：2014年07月21日)的优先权，其全部内容在此以援引的方式加入本说明书。
技术领域
本发明涉及基因工程领域，特别是涉及人FGFR2c胞外段146-356及其编码基因与应用。
背景技术
EGFR是原癌基因c-erbB1的表达产物，是表皮生长因子受体(HER)家族成员之一。该家族包括HER1(erbB1，EGFR)、HER2(erbB2，NEU)、HER3(erbB3)及HER4(erbB4)。HER家族在细胞生理过程中发挥重要的调节作用。EGFR信号通路对细胞的生长、增殖和分化等生理过程发挥重要的作用。EGFR等蛋白酪氨酸激酶功能缺失或其相关信号通路中关键因子的活性或细胞定位异常，均会引起恶性肿瘤、糖尿病、免疫缺陷及心血管疾病的发生。EGFR也是恶性肿瘤治疗中重要的分子靶点。
但是，对EGF信号的抑制往往导致FGF信号的激活，并产生耐药。因此，如果能够找到抑制EGF信号、但不激活FGF信号的蛋白质分子，则可以期待对恶性肿瘤有更好的治疗效果。
发明内容
本发明的目的是提供能够抑制EGF信号、但不激活FGF信号的蛋白质分子。
本发明人为解决上述现有技术中存在的技术问题进行了深入研究，结果发现：包含人FGFR2c胞外段第146-356位氨基酸序列的蛋白质及其变体能够抑制EGF信号、但不激活FGF信号，从而完成了本发明。
即，本发明包括：
1.一种分离的蛋白质，其包含下述氨基酸序列：
i).SEQ ID NO：1或2所述的氨基酸序列。
2.一种分离的蛋白质，其包含下述氨基酸序列，且具有抑制EGF信号、但不激活FGF信号的功能：
ii).在SEQ ID NO：1或2所述的氨基酸序列中缺失、取代、***和/或添加1个或多个氨基酸而得的氨基酸序列；
iii).与SEQ ID NO：1或2所述的氨基酸序列具有80％以上的氨基酸同源性的氨基酸序列；或者
vi).由在严格条件下与编码SEQ ID NO：1或2所述的氨基酸序列的核酸的互补链杂交的核酸编码的氨基酸序列。
3.根据项2所述的分离的蛋白质，其中，该分离的蛋白质来源于人。
4.编码项1～3中任一项所述的分离的蛋白质的分离的核酸。
5.一种含有项4所述核酸的载体。
6.一种含有项5所述载体的宿主细胞。
7.根据项6中所述的宿主细胞，其特征在于所述宿主细胞是CHO细胞、大肠杆菌细胞、昆虫细胞、酵母细胞中的任一种。
8.一种融合蛋白，其是项1～3中任一项所述的分离的蛋白质与其他多肽的融合蛋白。
9.根据项8所述的融合蛋白，其是项1～3中任一项所述的分离的蛋白质与人免疫球蛋白表位标记序列或人免疫球蛋白Fc段的融合蛋白。
10项1～3中任一项所述的分离的蛋白质、项4所述的核酸、项5所述的载体、项6或7所述的宿主细胞或项8或9所述的融合蛋白在制备用于治疗恶性肿瘤的药物中的用途。
11.一种治疗恶性肿瘤的药物组合物，其包含项1～3中任一项所述的分离的蛋白质、项4所述的核酸、项5所述的载体、项6或7所述的宿主细胞或项8或9所述的融合蛋白作为活性成分，以及药学上可接受的载体。
附图说明
图1是实施例1中人FGFR2c胞外段146-356(sFGFR2c)的SDS-PAGE图；其中，泳道M为蛋白Marker，泳道1为诱导的野生型(wsFGFR2c)，泳道2为未诱导的野生型，泳道3为诱导的S252W突变型(msFGFR2c)，泳道4为未诱导的S252W突变型。
图2是实施例1中sFGFR2c的蛋白western blot杂交图；其中，泳道1为野生型，泳道2为S252W突变型。
图3是实施例7中sFGFR2c(146-356)、sFGFR2c(147-366aa)和sFGFR2c(151-377aa)稳定性比较图和肿瘤细胞抑制效果比较图；其中图A是sFGFR2c稳定性的电泳图，图B是 sFGFR2c(147-366aa)稳定性的电泳图，图C是sFGFR2c(151-377aa)稳定性的电泳图。
图4是实施例8中sFGFR2c与EGFR的结合能力的co-IP图；其中，wsFGFR2c为野生型人FGFR2c胞外段146-356，msFGFR2c为S252W突变型FGFR2c胞外段146-356。
图5是实施例8中sFGFR2c对DU145细胞增殖影响的CCK8结果图；其中，wsFGFR2c为野生型人FGFR2c胞外段146-356，msFGFR2c为S252W突变型人FGFR2c胞外段146-356，▲代表与空白对照组相比，P＜0.01；★代表与EGF单独诱导组相比，P＜0.01。
图6是实施例8中人FGFR2c胞外段146-356对EGFR/ERK信号通路影响的蛋白杂交鉴定图；其中，wsFGFR2c为野生型人FGFR2c胞外段146-356，msFGFR2c为S252W突变型人FGFR2c胞外段146-356。
图7是通过western blot检测sFGFR2c抑制FGFRs和ERK磷酸化的实验结果图。
图8是通过等温滴定量热法(iTC)检测FGFR2c胞外段146-356与EGFR的相互作用的实验结果图。
具体实施方式
1.分离的蛋白质
在一个方面中，本发明提供一种分离的蛋白质，其包含下述氨基酸序列：
i).SEQ ID NO：1或2所述的氨基酸序列。
在本说明书中，术语“包含”优选意指“由……组成”。由SEQ ID NO：1所述的氨基酸序列组成的蛋白质是人FGFR2c胞外段的第146-356位氨基酸(wsFGFR2c)，由SEQ ID NO：2所述的氨基酸序列组成的蛋白质是在由SEQ ID NO：1所述的氨基酸序列组成的蛋白质中、对应于野生型人FGFR2c胞外段全长的第252位的丝氨酸(S)突变为色氨酸(W)而成的蛋白质(msFGFR2c)。在本说明书中，由SEQ ID NO：1或2所述的氨基酸序列组成的蛋白质统称为人FGFR2c胞外段146-356。
SEQ ID NO：1
Figure PCTCN2015084692-appb-000001
Figure PCTCN2015084692-appb-000002
SEQ ID NO：2
Figure PCTCN2015084692-appb-000003
成纤维细胞生长因子(Fibroblast Growth Factor，FGF)受体(FGF receptor，FGFR)是膜上受体，其胞外部分可与特异性配体结合，而胞内部分具有酪氨酸激酶活性，当胞外部分与配体结合后可激活受体的二聚化以及磷酸化，并导致下游信号的激活，从而调控靶基因的表达。FGFR2c胞外段是FGF受体2c亚型的胞外部分，可与配体结合，降低配体的有效浓度，从而抑制FGF信号。发明人已经验证，上述的分离的蛋白质能够抑制EGF信号、但不激活(优选抑制)FGF信号。
在其他方面中，本发明提供一种分离的蛋白质，其包含下述氨基酸序列，且具有抑制EGF信号、但不激活FGF信号的功能：
ii).在SEQ ID NO：1或2所述的氨基酸序列中缺失、取代、***和/或添加1个或多个氨基酸而得的氨基酸序列；
iii).与SEQ ID NO：1或2所述的氨基酸序列具有80％以上、优选85％以上、更优选87.8％ 以上、更优选90％以上、更优选95％以上、更优96％以上、更优选97％以上、更优选98％以上、更优选99％以上、更优选99.5％以上的氨基酸同源性的氨基酸序列；或者
vi).由在严格条件下与编码SEQ ID NO：1或2所述的氨基酸序列的核酸的互补链杂交的核酸编码的氨基酸序列。
在本说明书中，将上述的分离的蛋白质称为人FGFR2c胞外段146-356的变体。
优选地，所述人FGFR2c胞外段146-356的变体来源于人。
所述人FGFR2c胞外段146-356的变体中，氨基酸取代可以是保守取代，即将特定的氨基酸残基替换为具有相似物理化学特征的残基。保守取代的非限定性例子包括含脂肪族基团氨基酸残基之间的取代(例如Ile、Val、Leu或Ala间的相互取代)、极性残基之间的取代(例如Lys和Arg、Glu和Asp、Gln和Asn之间的相互取代)等。氨基酸缺失、取代、***和/或添加而成的变体可以通过对编码野生型蛋白质的DNA实施例如公知的定点诱变(例如参见Nucleic Acid Research,Vol.10,No.20,p.6487-6500,1982,通过引用其全文引入本说明书)或者人工合成蛋白质的方法来产生。在本说明书中，“一个或多个氨基酸”指通过定点诱变或人工合成方法能够缺失、取代、***和/或添加的程度的氨基酸，例如1～20个氨基酸、优选1～15个氨基酸、更优选1～10个氨基酸、更优选1～8个氨基酸、更优选1～2个氨基酸、更优选1个氨基酸。
两个氨基酸序列的同源性％可通过目测和数学计算来确定。或者两个多肽序列的同源性百分比可以通过使用以Needleman,S.B.及Wunsch,C.D.(J.Mol.Bol.,48:443-453,1970)的算法为基础的、可从威斯康星州大学遗传学计算机组(UWGCG)获得的GAP计算机程序进行序列信息比较来决定。GAP程序优选的默认参数包括：(1)Henikoff,S.以及Henikoff,J.G.(Proc.Natl.Acad.Sci.USA,89:10915-10919,1992)所记载的得分/矩阵、blosum62；(2)一个空位扣12分；(3)连续空位加扣4分；以及(4)末端空位不扣分。也可使用该领域技术人员使用的进行序列比较的其它程序。关于同源性百分比，例如：Altschul等(Nucl.Acids.Res.,25,p.3389-3402,1997)中记载的可用BLAST程序对序列信息进行比较和确定。该程序可于网络上在NantionalCenter for Biotechnology Information(NCBI)或DNA Data Bank of Japan(DDBJ)网站使用。在相同站点，对利用BLAST程序进行同源性检索的各种条件(参数)进行了详细记载，可对部分设定进行适当变更，但检索通常用默认值进行。此外，两个氨基酸序列的同源性％也可用遗传信息处理软件GENETYX Ver.7(GENETYX制)等程序或FASTA算法等来确定。此时，也可用默认值检索。
本说明书中，“严格条件”是指形成所谓的特异性杂合体并且不形成非特异性杂合体的条 件。严格条件的实例包括如下的条件，在该条件下，高度同源的DNA彼此杂交，例如，不低于80％同源的、优选不低于90％同源的、更优选不低于95％同源的、仍更优选不低于97％同源的、特别优选不低于99％同源的DNA彼此杂交，并且同源性比上述低的DNA彼此不杂交，或者典型Southern杂交的洗涤条件，即，在对应于1xSSC，0.1％SDS在60℃、优选0.1xSSC，0.1％SDS在60℃、更优选0.1xSSC，0.1％SDS在68℃的盐浓度和温度下洗涤1次、优选2或3次的条件。
是否具有“抑制EGF信号、但不激活(优选抑制)FGF信号”的功能可以采用例如实施例中所描述的方法来确定。
2.分离的核酸
在其他方面中，本发明提供一种分离的核酸，其编码上述人FGFR2c胞外段146-356或其变体。
所述分离的核酸可以是单链，也可以是双链；可以是DNA，也可以是RNA，还可以是DNA与RNA的杂交体。
所述分离的核酸可以采用人工合成方法来制备，也可以例如采用基因工程方法来制备。
典型地，上述SEQ ID NO：1或2所述的氨基酸序列可以分别由SEQ ID NO：3或4所述的碱基序列编码。
3.载体和宿主细胞
在其他方面中，本发明提供包含所述核酸的载体。
对于载体的类型没有特殊限制，可以是本领域技术人员常规使用的那些。作为载体，可以列举出质粒、噬菌体、动物病毒等。优选地，所述载体是表达载体。所述的表达载体包括原核表达载体和真核表达载体，优选为pET3c载体、pCDNA3.1载体、pIRESneo3载体、pPICZαA载体或pFastBac载体。
在其他方面中，本发明提供包含所述载体的宿主细胞。
对于宿主细胞的类型没有特殊限制，可以是本领域技术人员常规使用的那些。作为宿主细胞，可以列举出CHO细胞、大肠杆菌细胞、昆虫细胞、酵母细胞等。
4.融合蛋白
在其他方面中，本发明提供上述的人FGFR2c胞外段146-356或其变体与其他多肽的融 合蛋白。优选地，所述其他多肽是人免疫球蛋白表位标记序列或人免疫球蛋白Fc段。
所述融合蛋白可以采用本技术领域的常规方法来制备。
5.药物组合物及制药用途
在其他方面中，本发明提供一种药物组合物，其包含上述的人FGFR2c胞外段146-356或其变体、上述核酸、上述的载体、上述的宿主细胞或上述的融合蛋白作为活性成分，还包含药学上可接受的载体。本发明的药物组合物可用于治疗恶性肿瘤。因此，在其他方面中，本发明提供上述的人FGFR2c胞外段146-356或其变体、上述核酸、上述的载体、上述的宿主细胞或上述的融合蛋白在制备用于治疗恶性肿瘤中的用途。
作为恶性肿瘤，可以列举出***癌、口腔癌、鼻粘膜癌，气管癌、支气管癌、肺癌、食管癌，胃癌、大肠癌、小肠癌、肝癌、胆管癌，胆囊癌，胰腺癌、肾癌、膀胱癌、尿道癌、睾癌、卵巢癌、甲状腺癌、肾上腺癌、胸腺癌、***癌、乳腺癌、扁桃腺癌等。作为药学上可接受的载体，具体地可以列举出无菌水、生理盐水、植物油、乳化剂、悬浮剂、表面活性剂、稳定剂、增味剂、赋形剂、载体、防腐剂、结合剂等。
实施例
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。所用到的细胞株和载体均为商业化产品。F开头的引物一般为上游引物，R开头的引物一般为下游引物。
实施例1 野生型及突变型FGFR2c胞外段146-356多肽基因在大肠杆菌中的表达
本实施例描述野生型wsFGFR2c和S252W突变型msFGFR2c的制备过程。
一、FGFR2c胞外段146-356多肽基因的制备：
1、用Trizol法从人胎盘组织(从广东某医院经产妇同意获得新鲜胎盘组织)提取野生型FGFR2c胞外段总RNA，并建立cDNA文库；
(1)用Trizol法抽提mRNA：使用Trizol试剂进行抽提，具体步骤见文献“张志成.突变型βFGFR2IIIc胞外段的原核表达、体外复性及活性研究.暨南大学硕士论文,2007:46-47”。
(2)进行RT-PCR以获得cDNA：
计算好整个逆转录PCR各成分的用量，先在PCR管内加入步骤(1)制备的样品总RNA和DEPC处理水，在PCR仪上进行RNA预变性，条件为65℃，10min。预变性后立即***冰中。然后再添加逆转录反应中的其他成分，进行逆转录反应，得到cDNA。具体如下：
Figure PCTCN2015084692-appb-000004
Figure PCTCN2015084692-appb-000005
逆转录PCR程序：30℃，10min；42℃，1h；70℃，10min。
2、引物的设计：
本发明中引物合成均来自于北京六合华大基因科技股份有限公司。
本发明中限制性内切酶均来自于TaKaRa。
野生型wsFGFR2c基因引物序列如下：
F1：5’-CG CATATG AACAAGAGAGCACCATAC-3’；划横线为Nde Ⅰ酶切位点；
R1：5’-AT GGATCC CTATTA CAGAACTGTCAACCATGC-3’；划横线为BamH I酶切位点。
为获得编码S252W突变型msFGFR2c的基因，设计一对定点突变引物，如下：
F2：5’-TTGTGGAGCGATGGCCTCACCGGCCCAT-3’；
R2：5’-ATGGGCCGGTGAGGCCATCGCTCCACAA-3’。
3、FGFR2c胞外段146-356基因序列的扩增：
为扩增野生型FGFR2c胞外段146-356基因，使用野生型wsFGFR2c引物(F1和R1)对步骤1得到的cDNA进行PCR，得到野生型wsFGFR2c基因。
为扩增突变型FGFR2c胞外段146-356基因，利用重叠延伸PCR法，以野生型wsFGFR2c基因为模板，分别使用突变型PCR引物，扩增得到S252W突变型msFGFR2c基因。
上述PCR的扩增体系和反应条件如下(PrimerSTAR max来自TaKaRa)：
野生型FGFR2c PCR反应体系：
Figure PCTCN2015084692-appb-000006
重叠PCR进行点突变获得S252W突变型msFGFR2c基因包括两个步骤：
第一步PCR反应体系：
Figure PCTCN2015084692-appb-000007
Figure PCTCN2015084692-appb-000008
第二步PCR反应体系：
Figure PCTCN2015084692-appb-000009
反应条件为：96℃5min；94℃15s、60℃15s、72℃5s，31个循环；72℃10min。
4、收集、纯化、鉴定步骤：
1％浓度的琼脂糖电泳，切胶，使用DNA凝胶回收试剂盒(TIANGEN，DP209)回收DNA，回收得到的DNA片段可用琼脂糖凝胶电泳和紫外分光光度计检测浓度和纯度。OD260/OD280比值应为1.7-1.9。
二、野生型及突变型的FGFR2c胞外段146-356多肽基因重组载体的构建：
1、以双酶切和连接反应构建重组质粒
分别将PCR扩增的序列与pET3c载体(来自美国Invitrogen公司)同时进行Nde Ⅰ和BamH Ⅰ双酶切，酶切反应条件：37℃水浴4h；
pET3c质粒和FGFR2c基因分别经Nde Ⅰ和BamH Ⅰ双酶切后，使用T4DNA连接酶进行连接，按T4DNA连接酶说明书操作，配制反应体系，连接反应条件为16℃水浴12h，得到连接产物。
2、重组质粒的表达和鉴定
I、CaCl2法转化大肠杆菌DH5α菌株：先用CaCl2制备大肠杆菌DH5α感受态，接着使用上述步骤得到的连接产物转化大肠杆菌DH5α感受态，具体步骤见文献“张志成.突变型βFGFR2IIIc胞外段的原核表达、体外复性及活性研究.暨南大学硕士论文,2007:46”。
II、重组质粒的双酶切鉴定：
用接种环多次挑取单菌落分别至于含100μg/ml氨苄青霉素(Amp)的5ml LB培养基中，并做好标记，37℃摇床震荡培养12h，抽提质粒(按照OMEGA质粒小提试剂盒操作方法)，进行双酶切鉴定(使用Nde Ⅰ和BamH Ⅰ双酶切)，选取双酶切鉴定成功的单克隆送生工生物公司测序，获得测序正确的pET3c-FGFR2c、pET3c-S252W-FGFR2c两种重组质粒。
三、FGFR2c胞外段146-356多肽基因在大肠杆菌中的表达：
(1)按照上述CaCl2法转化大肠杆菌DH5α菌株的方法，同理将上述两种重组质粒分别转入到大肠杆菌BL21(DE3)(Novagen)工程菌种中，获得表达菌株；
(2)按体积比1：50的接种比例将BL21(DE3)表达菌株接种到含有质量体积比0.1％Amp的灭菌的LB液体培养基中，37℃、200rpm摇床培养。
当菌体OD600达到0.6～0.8时，设对照组和诱导组：诱导组加0.84M IPTG至终浓度为0.84mM，37℃诱导表达3h；对照组不做任何处理。
以SDS-PAGE电泳鉴定目的蛋白表达情况，结果如图1所示。结果表明，IPTG能较好地诱导BL21(DE3)表达菌株表达FGFR2c胞外段146-356。
四、FGFR2c胞外段146-356多肽的收集与纯化：
(1)按步骤三的方法获得IPTG诱导后的菌体，接着4℃、6000rpm离心30min收集菌体沉淀。
(2)按菌体：破碎缓冲液＝1g：(8～10)ml的比例，超声破碎菌体，破碎完成后4℃、18000rpm离心60min，收集破碎的上清。破碎条件是：工作3s，停5s，破碎时间为18min，振幅为65％，破碎缓冲液为：0.15M NaCl、25mM磷酸钠缓冲液(PB，磷酸二氢钠和磷酸氢二钠搭配得到)、2mM EDTA，pH＝7.5。
(3)在一些原核表达的菌中，FGFR2c胞外段146-356蛋白以包涵体形式表达，则通过包涵体清洗以及变复性技术来获得活性形式的FGFR2c胞外段146-356蛋白(具体方法可参考专利ZL200710029286.6实施例1)。
(4)通过Western Blot进行检测，一抗为Bek抗体(c-17)(santa Cruz Blotechnology)，二抗为兔二抗(cat NO.AS006,Asbio)。结果如图2所示，表明FGFR2c胞外段146-356能被FGFR抗体特异性识别。
实施例2 FGFR2c胞外段146-356在哺乳动物细胞中表达
该实施例描述通过在哺乳动物细胞中的重组表达野生型wsFGFR2c、S252W突变型msFGFR2c基因制备潜在糖基化形式的FGFRc胞外段146-356多肽的方法。
1、引物设计如下：
F3： 5’-ATAT GGATCC GCCGCCACC ATG AACAAGAGAGCACCATAC-3’；划横线为BamH I酶切位点；
R3： 5’-GCGCAAGCTT TCATTA CAGAACTGTCAACCATGC-3’；划横线为HindⅢ酶切位点。
2、载体构建：
载体pCDNA3.1(-)(购买于美国Invitrogen公司)作为表达载体；
以实施例1得到的pET3c-FGFR2c、pET3c-S252W-FGFR2c为模板，以F3和R3为引物，进行PCR，反应体系和条件同实施例1。将得到的野生型wsFGFR2c基因、S252W突变型msFGFR2c基因分别连接到pCDNA3.1(-)，转化大肠杆菌DH5α感受态细胞，由此产生的载体称为pCDNA3.1-FGFR2c、pCDNA3.1-FGFR2c-S252W、(具体步骤同实施例1，所用的双酶切的酶为BamH I和Hind Ⅲ)。
3、pCDNA3.1-FGFR2c、pCDNA3.1-FGFR2c-S252W分别转染293细胞
(1)按照去内毒素质粒小提试剂盒的方法(购买于OMEGA公司)抽提质粒，得到pCDNA3.1-FGFR2c、pCDNA3.1-FGFR2c-S252W质粒；
(2)选用人胚肾细胞293T细胞(ATCC，CRL-3216)为宿主细胞，以添加了胎牛血清(FBS，终浓度为10％v/v)的DMEM培养基，37℃、5％CO2饱和湿度培养箱培养细胞生长至融合；
(3)转染24h前，用质量体积比0.25％的胰酶消化对数期生长的293T细胞，用无双抗的DMEM培养基吹打细胞成悬液，铺于六孔板中，每孔2×105个，使其在转染时完全贴壁，且细胞密度达到50％-60％；
(4)用LipofectamineTM2000脂质体转染试剂盒(购买于美国Invirtogen公司)进行转染，具体转染步骤如下：
①LipofectamineTM2000的稀释：按六孔板每孔的量计算：5μL LipofectamineTM2000加到250μL opti-MEM培养基的比例稀释，静置5min；
②待转染质粒的稀释：按六孔板每孔的量计算：4μg待转染质粒加到250μL opti-MEM培养基的比例稀释；
③将步骤①和步骤②得到的稀释液等体积混合，静置20min，每孔加500μL混合液，每孔补加1.5ml opti-MEM培养基；37℃、5％CO2的培养箱培养4-6h后换成含体积百分比10％FBS的DMEM培养基；转染24h后，小心吸去旧的培养基，换上含10％(v/v)FBS的DMEM完全培养基，继续培养。
4、FGFR2c胞外段146-356蛋白质的纯化与鉴定：
4℃、18000rpm离心30min收集培养液上清，蛋白质纯化步骤为：
使用肝素亲和层析柱(GE 17-0998-01 50ml)，用双蒸水冲洗柱子3个柱体积后用亲和层析平衡液平衡柱子，流速为5ml/min，平衡至少3个柱体积后将步骤(2)得到的上清上样，上样完成后继续用亲和层析平衡液冲洗3个柱体积，换成肝素洗脱液进行洗脱，在波长为280nm处收集单一的洗脱峰，得到野生型wsFGFR2c多肽和S252W突变型msFGFR2c多肽，-70℃保存，用于后续试验。
亲和层析平衡液：25mM HEPES、0.15M NaCl，pH＝7.5；
肝素洗脱液：25mM HEPES、1.5M NaCl，pH＝7.5；
流速为5ml/min。
通过Western Blot进行鉴定。结果表明FGFR2c胞外段146-356能被FGFR抗体特异性识别。
实施例3 FGFR2c胞外段146-356在CHO细胞中表达
1、引物设计：
上游引物F3：5’-ATAT GGATCC GCCGCCACC ATG AACAAGAGAGCACCATAC-3’；划横线为BamH I酶切位点；
下游引物R4：5’-GCGCGAATTC TCATTA CAGAACTGTCAACCATGC-3’划横线为EcoR I 酶切位点。
2、载体构建：
载体pIRESneo3(购自Clontech公司)作为表达载体；
按实施例2步骤2操作，所用的引物为F3和R4，所用的限制性内切酶为BamH I和EcoR I。由此产生的载体称为pIRESneo3-FGFR2c、pIRESneo3-FGFR2c-S252W。
3、pIRESneo3-FGFR2c、pIRESneo3-FGFR2c-S252W分别转染CHO-DG44细胞
(1)用无内毒素质粒大提试剂盒抽提质粒，得到pIRESneo3-FGFR2c、pIRESneo3-FGFR2c-S252W质粒；
(2)分别用步骤(1)得到的质粒转染中华仓鼠卵巢细胞CHO-DG44细胞(Invitrogen公司)，转染步骤同实施例2；经嘌呤霉素高压(400ng/ml)筛选后得到稳定克隆的重组CHO细胞；
(3)取重组CHO细胞，按5×105个/ml接种于1.5L含4mmol/L谷氨酰胺、0.68mg/L次黄嘌呤、0.194mg/L胸腺嘧啶的proCHO5培养基中，用5L摇瓶培养，在转速为110r/min的条件下，37℃培养72h后，转至31℃继续培养216h；
(4)收获1.5L培养体积下细胞培养液上清，取500ml经0.45μm滤膜过滤，按照实施例2的方法使用肝素亲和层析柱纯化目的蛋白，并通过Western Blot进行鉴定。结果表明FGFR2c胞外段146-356能被FGFR抗体特异性识别。
实施例4 FGFR2c胞外段146-356多肽在酵母细胞中表达
1、引物设计：
上游引物F5：5’-ATAT CTCGAG GCCGCCACC ATG AACAAGAGAGCACCATAC-3’；划横线为Xho I酶切位点；
下游引物R5：5’-GCGC TCTAGA TCATTA CAGAACTGTCAACCATGC-3’；划横线为Xba Ⅰ酶切位点。
2、载体的构建：
所用的载体为毕赤酵母表达载体pPICZαA(Invitrogen)。
按实施例2步骤2操作，所用的引物为F5和R5，所用的酶为Xho I和Xba Ⅰ。由此产生的载体称为pPICZαA-FGFR2c、pPICZαA-FGFR2c-S252W。
3、酵母细胞的转化及鉴定：
按照实施例1中CaCl2法转化大肠杆菌DH5α菌株的方法，将pPICZαA-FGFR2c和pPICZαA-FGFR2c-S252W分别转入大肠杆菌DH5α菌中。在含Zeocin(100ug/ml)抗生素的LB平板上对转化的DH5α进行初步筛选，挑平板上生长的单菌落在37℃、220rpm振荡培养16h，小量提质粒及酶切鉴定(使用Xho I和Xba Ⅰ进行双酶切鉴定)，挑选阳性克隆送至公司测序。
4、酵母细胞X33的转化：
将提纯的质粒用Sac I酶酶切线性化后(酶切体系是10×buffer 2μl、质粒10μl、Sac I 1μl， 加ddH20至20μl)，分别电转化毕赤酵母X33感受态细胞，具体如下：取80μl X33感受态细胞和20μl线性化的质粒混匀后转入预冷的0.2cm间隙的电击杯中，冰浴5min；电脉冲能转化电压1800V、4.3ms电击转化；立即加入1mL预冷的1M山梨醇至杯中，用移液器轻轻混匀后转到1.5mL EP管中，涂布于含有100ug/ml Zeocin的高渗完全培养基(YPDS)平板，28℃培养2～3d。
5、重组菌株的鉴定和诱导表达：具体步骤参见文献：王丁丁等.一种融合抗体ScFv-Fc通用表达载体的构建.中国生物工程杂志.2011,31(8):110-117中的融合抗体ScFv-Fc的表达。
6、FGFR2c胞外段146-356蛋白质的纯化与鉴定
(1)通过离心从发酵培养基中除去酵母细胞；
(2)使用8000KD的柱式超滤器浓缩培养基，初步分离和纯化重组FGFR2c胞外段146-356；
(3)使用肝素柱亲和层析(同实施例2)，进一步纯化含FGFR2c胞外段146-356的浓缩物，并通过Western Blot进行鉴定。结果表明FGFR2c胞外段146-356能被FGFR抗体特异性识别。
实施例5 FGFR2c胞外段146-356在杆状病毒及感染的昆虫细胞中的表达
1、按照实施例3的方法获得载体pFastBac-FGFR2c和pFastBac-FGFR2c-S252W(引物同实施例3，pFastBac载体来自Invitrogen)。
2、转化大肠杆菌DH10Bac感受态
大肠杆菌感受态DH10Bac(来自Invitrogen)从-80℃中取出后放到冰上溶解；取5μL质粒在无菌条件下加到大肠杆菌感受态DH10Bac中，冰上放置30min，42℃热激45s，热激后立刻放到冰上，静置2分钟；加0.9ml室温的S.O.C.培养基(Cat.No.15544-034)，225rpm(37℃)震荡45min；用S.O.C.培养基按体积比1：10比例稀释培养物，取100μL稀释液涂到含有50μg/ml卡那(kanamycin)、10μg/ml四环素(tetracycline)、7μg/ml庆大霉素(gentamicin)、200mg/ml IPTG和20mg/mL X-gal的LB平板上，将涂好的平板放到37℃培养24h；次日，挑取白斑在含有50μg/ml kana的LB液体培养基上培养，菌液进行PCR鉴定；将鉴定正确的菌液接种到5ml培养基中，37℃培养过夜；提取阳性杆粒。利用Bac-to-bac HT Vector kit(购自invitrogen公司，cat.1058-027)试剂盒抽提重组质粒DNA，琼脂糖电泳检测转化结果。
3、重组杆状病毒pFastBac载体的转染的具体方法见文献“谢秋玲等.重组人可溶性PDGFRβ/Fc在昆虫细胞Sf9中的表达.昆虫学报.July 2009,52(7):743-748”。
4、FGFR2c胞外段表达：
收集上清和细胞，16000rpm、4℃离心30min后对上清及细胞沉淀进行SDS-PAGE电泳，并通过Western Blot进行鉴定。结果表明FGFR2c胞外段146-356能被FGFR抗体特异性识别。
实施例6 FGFR2c胞外段146-356-Fc段融合蛋白在酵母细胞的表达
1、Fc段基因的获取
(1)引物设计：
F9-Fc：
5’-CCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGTCCTGCTCCAGAACTCCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTG-3’；
F8-Fc：
5’-AAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCACAGGTCAAGTTCAACTGGTACGTGGAC-3’；
F7-Fc：
5’-TTCAACTGGTACGTGGACGGCGTGCAGGTGCACAACGCCAAGACCAAGCCCCGGGAGCAGCAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTG-3’；
F6-Fc：
5’-TCCGTGCTGACCGTGCTGCACCAGAACTGGCTGGACGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATC GAGAAAACCATCAGCAAG-3’；
R6-Fc：
5’-CAGGGACACCTGGTTCTTGGTCATTTCCTCTCGAGAGGGTGGCAGGGTGTACACCTGGGGTTCCCTGGGCTGGCCCTTGGCCTTGCTGATGGTTTTCTC-3’；
R7-Fc：
5’-TCTTGTAGTTGTTCTCGGGCTGGCCGTTGCTCTCCCACTCCACGGCGATGTCGCTGGGGTAGAAGCCCTTCACCAGGCAGGTCAGGGACACCTGGTTCTT-3’；
R8-Fc:
5’-CCCTGCTGCCATCTGCTCTTGTCCACGGTCAGCTTGCTGTACAGGAAGAAGCTGCCGTCGCTGTCCAGCACTGGGGGGGTGGTCTTGTAGTTGTTCTCGG-3’；
R9-Fc:
5’-CTTGCCGGGGGACAGGCTCAGGCTCTTCTGGGTGTAGTGGTTGTGCAGGGCCTCGTGCATCACGCTGCAGCTGAACACGTTGCCCTGCTGCCATCTGCTC-3’。
(2)经4步PCR获得Fc段目的基因，第一步是以F6-Fc和R6-Fc为引物进行PCR，1％浓度的琼脂糖电泳，切胶回收(TIANGEN，DP209)；第二步以F7-Fc和R7-Fc为引物，第一步PCR产物为模板，进行PCR，然后切胶回收；第三步以F8-Fc和R8-Fc为引物，第二步PCR产物为模板，进行PCR，然后切胶回收；第四步以F9-Fc和R9-Fc为引物，第三步PCR产物为模板，进行PCR，然后切胶回收；
PCR反应体系：
Figure PCTCN2015084692-appb-000010
2、通过PCR和重叠PCR分别扩增获得FGFR2c胞外段、Fc段和FGFR2c-L-Fc目的基因。
以pET3c-FGFR2c质粒段基因为模板，F10-FGFR2c和R10-FGFR2c为引物，PCR得到 FGFR2c模板片段；以Fc段基因为模板，F11-Fc和R11-Fc为引物，PCR得到Fc段模板片段。
(1)引物设计如下：均为5’-3’
F10-FGFR2c：ATATACTAGT ATG GATGACGACGACAAG AACAAGAGAGCACCATAC；划横线为SpeI酶切位点；
R10-FGFR2c：CGACCCACCACCGCCGGAGCCACCGCCACC CAGAACTGTCAACCATGC；
F11-Fc：GGCGGTGGTGGGTCGGGTGGCGGCGGATCTCCCAAGAGCTGCGACAAG；
R11-Fc：ATATGAATTCTCATTACTTGCCGGGGGACAGG；划横线为EcoR I酶切位点；
(2)PCR反应体系和反应条件如下：
PCR反应体系
Figure PCTCN2015084692-appb-000011
反应条件为：96℃5min；94℃15s、60℃15s、72℃5s，31个循环；72℃10min。
(3)按照实施例1中PCR产物切胶回收的方法回收FGFR2c和Fc段DNA：
重叠PCR反应体系：
Figure PCTCN2015084692-appb-000012
反应条件为：96℃5min；94℃15s、60℃15s、72℃5s，5个循环；72℃10min。
(4)上述5个循环完成后加F10-FGFR2c和R11-Fc引物各3μL，再进行30个循环，PCR产物切胶回收的方法按照实施例1，得到FGFR2c-L-Fc基因。
3、按照实施例4的方法，将FGFR2c-L-Fc基因构建到pPICZαA表达载体，并以双酶切鉴定和测序结果显示成功构建FGFR2c-L-Fc-pPICZαA重组质粒；
4、按照实施例4的方法，诱导表达以及纯化鉴定融合蛋白FGFR2b-L-Fc，结果表明融合蛋白FGFR2b-L-Fc能被FGFR抗体特异性识别。
实施例7稳定性比较
按文献(刘雪婷.野生型和突变型FGFR2Ⅲc胞外段的原核表达及其对肿瘤的抑制作用.暨南大学硕士论文.2008：8)中的方法制备得到S252W突变型msFGFR2c(147-366)和野生型FGFR2c(147-366)，以及按文献(何颖，汪炬等.FGFR2IIIc胞外段的原核表达及其对***癌细胞增殖的抑制作用.中国生物工程杂志.2009,29(7):7～11)中的方法制备得到 S252W突变型msFGFR2c(151-377)和野生型FGFR2c(151-377)。
1、SDS-PAGE电泳检测sFGFR2c稳定性
将S252W突变型msFGFR2c多肽(实施例1制备得到)和野生型wsFGFR2c多肽(实施例1制备得到)，S252W突变型msFGFR2c(147-366)、野生型FGFR2c(147-366)，S252W突变型msFGFR2c(151-377)、野生型FGFR2c(151-377)分别通过25mM HEPES缓冲液透析12h，接着用0.22μm的滤膜过膜，再用25mM HEPES缓冲液稀释至蛋白浓度均为100μg/ml。各取2ml置于4℃冰箱，第1、3、5、7天同一时刻各取上清160μl(每次取样的时候都需4℃、16000rpm离心15min，弃沉淀)，将取样的样品置于-70℃。然后SDS-PAGE电泳检测蛋白质稳定情况。同时，用透析过膜的蛋白溶液作为对照。
2、结果
实验结果如图3所示：图3A为本发明制备的FGFR2c胞外段146-356的稳定性电泳图，泳道1是作为对照的S252W突变型msFGFR2c，泳道2、3、4、5分别是S252W突变型msFGFR2c第1、3、5、7天样品；泳道6是作为对照的野生型wsFGFR2c，泳道7、8、9、10分别是野生型wsFGFR2c第1、3、5、7天样品。从图中可以看出本发明制备的FGFR2胞外段基本上没有降解，蛋白比较稳定。
图3B为按文献制备的S252W突变型msFGFR2c(147-366aa)和野生型FGFR2c(147-366aa)的稳定性电泳图，泳道1是作为对照的S252W突变型msFGFR2c(147-366)，泳道2、3、4、5分别是S252W突变型msFGFR2c(147-366)第1、3、5、7天样品；泳道6是作为对照的野生型FGFR2c(147-366)，泳道7、8、9、10分别是野生型FGFR2c(147-366)第1、3、5、7天样品。从图中看出，野生型和S252W突变在第1、3天有少量降解出现(泳道2、3、7、8)，第5天进一步降解(泳道4、9)，到第7天的时候基本上降解完全(泳道5、10)。
图3C为按文献制备的S252W突变型msFGFR2c(151-377aa)和野生型FGFR2c(151-377aa)的稳定性电泳图，泳道1是作为对照的S252W突变型msFGFR2c(151-377aa)，泳道2、3、4、5分别是S252W突变型msFGFR2c(151-377aa)第1、3、5、7天样品；泳道6是作为对照的野生型FGFR2c(151-377aa)，泳道7、8、9、10分别是野生型FGFR2c(151-377aa)第1、3、5、7天样品。从图中看出，野生型和S252W突变在第1、3天有降解出现(泳道2、3、7、8)，第5天进一步降解(泳道4、9)，到第7天的时候基本上降解完全(泳道5、10)。
结论：本发明构建的FGFR2c胞外段146-356比现有技术FGFR2c胞外段(147-366aa)、FGFR2c胞外段(151-377aa)更稳定，有利于提高工艺的得率。
实施例8 FGFR2c胞外段146-356通过抑制DU145细胞EGF信号抑制恶性肿瘤
1、细胞培养
***癌DU145细胞(来自ATCC)传代培养于50cm2细胞培养瓶(购买于Thermo)中，添加含有10％(v/v)胎牛血清的1640培养基，于37℃、5％CO2的细胞培养箱中培养。培养过程中，当细胞间的汇合程度达80％左右时即可进行传代，使用质量体积比0.25％胰蛋白酶液消化细胞，正常传代时细胞的密度至少达5×105个/mL。
2、免疫共沉淀(Co-IP)检测sFGFR2c与外源和内源EGFR结合
(1)人胚肾细胞293T细胞(ATCC，CRL-3216)过表达EGFR
1)转染和诱导：
①转染：具体方法同实施例2 lipofectamineTM2000脂质体转染
②诱导：培养24后用1×PBS洗涤去残余培养基，换含有0.5％(v/v)FBS的DMEM的饥饿培养基，12h后进行诱导。具体步骤：将含有EGF(20ng/ml)或FGF-2(20ng/ml)或wsFGFR2c(1μg/ml，实施例1制备)或msFGFR2c(1μg/ml，实施例1制备)的3％(v/v)FBS的DMEM培养基加入6孔板中，每孔2ml；第1个孔只加3％(v/v)FBS的DMEM培养基，第2个孔加3％(v/v)FBS的DMEM培养基+wsFGFR2c，第3个孔加3％(v/v)FBS的DMEM培养基+wsFGFR2c+EGF，第4个孔加3％(v/v)FBS的DMEM培养基+wsFGFR2c+FGF-2，第5个孔加3％(v/v)FBS的DMEM培养基+msFGFR2c，第6个孔加3％(v/v)FBS的DMEM培养基+msFGFR2c+EGF，第7个孔加3％(v/v)FBS的DMEM培养基+msFGFR2c+FGF-2。
2)Co-IP制样
I、细胞裂解制样
①诱导1h后，先用冰预冷的1×PBS洗两次，把1×PBS彻底吸干。
②将细胞裂解液(碧云天,货号P0013)加入步骤①的细胞中，每孔加入400μL细胞裂解液。
③用细胞刮把所有细胞刮下并收集到EP管。
④振荡器上振荡30s，冰上静置裂解10min，之后4℃、16000rpm离心10min；弃沉淀，取上清，得到细胞裂解液蛋白样品。
II、孵育磁珠、制样
①取磁珠(dynabeadsM-270streptavidin)加入EP管，40μL/管；每管加入600μL 1×PBS，重复洗三次。
②去除PBS，每管加入细胞裂解液蛋白样品300μL，与磁珠孵育1h。
③孵育完毕后，用含0.05％(v/v)吐温的PBS清洗磁珠，5min换一次液，重复8次。之后再用PBS洗2次，每次5min。
④吸干PBS，加入5×SDS上样buffer，沸水浴5min，制成Western blot样品。
⑤进行western blot检测，结果如图4所示。
3、CCK8法检测细胞增殖
(1)铺板：DU145细胞传代后长至七到八成满，用胰酶消化，加入含10％(v/v)FBS的1640培养基把细胞稀释至3×104个/mL的细胞悬浮液，再按(4～5)×103个细胞/孔的量加入到96孔细胞培养板中，即每孔加150μL细胞悬浮液，37℃、5％CO2培养箱中培养24h。
(2)饥饿：吸去原培养基，加入含0.1％活性炭处理血清的1640培养基150μL/孔，饥饿培养24h。
(3)诱导：吸去饥饿培养基，重新加入含0.1％活性炭处理血清的1640培养基，加入 sFGFR2c进行诱导150μL/孔；其诱导过程为：96孔板取7列，第1列只加150μl培养基，第2列加培养基+EGF，第3列加培养基+EGF+sFGFR2c(40ng/ml)，第4列加培养基+EGF+sFGFR2c(80ng/ml)，第5列加培养基+EGF+sFGFR2c(160ng/ml)，第6列加培养基+EGF+sFGFR2c(320ng/ml)，第7列加培养基+EGF+sFGFR2c(640ng/ml)，诱导培养48h。
(4)读板：先按CCK8说明书配制好CCK8工作液，再吸干每孔的培养基的溶液，加入CCK8工作液110μL/孔，培养箱中37℃孵育4h，取出轻轻振荡，在酶标仪中读450/570nm双波长吸光值。
4、实验结果
(1)Co-IP检测EGFR与sFGFR2c结合(见图4)
Co-IP实验检测DU145细胞内源表达的EGFR与sFGFR2c的结合能力。结果发现，当有EGF存在的情况下，msFGFR2c(即S252W突变型msFGFR2c，下同)与EGFR的结合力减弱(见图3B)，同样，wsFGFR2c(即野生型wsFGFR2c)的情况也一样。另外，我们还发现，同时加入FGF-2和sFGFR2诱导DU145细胞也能引起EGFR与sFGFR2c相互结合，但这种结合远比sFGFR2c单独诱导或与EGF共同诱导时的小。
(2)EGF诱导DU145细胞增殖，同时加入sFGFR2c进行诱导时，对DU145的增殖具有抑制作用(见图5)。当EGF浓度为5ng/mL时，随着sFGFR2c浓度的增大，对EGF诱导的增殖作用抑制效果越明显，尤其是msFGFR2c，当浓度达160ng/mL时抑制作用最好，相对于对照组抑制率达21.1％，当msFGFR2c诱导浓度继续增大时，其抑制效果有所下降。而野生型wsFGFR2c的抑制效果相对较弱，在320ng/mL时相对于对照组其相对生长率为102.5％，同样，诱导浓度继续增大时，其抑制作用也有所减弱。
(3)sFGFR2c抑制EGFR/ERK信号通路(见图6)
通过western blot检测发现用sFGFR2c诱导DU145细胞后，sFGFR2c能抑制EGF诱导的EGFR和ERK磷酸化(见图6)，在与EGF共同诱导时二者都能抑制EGFR和ERK的激活，且msFGFR2c较wsFGFR2c的抑制作用更强。类似地，sFGFR2c也能抑制由FGF-2诱导的ERK磷酸化。
(4)sFGFR2c抑制FGF信号通路
通过western blot检测发现用sFGFR2c诱导DU145细胞后，sFGFR2c能抑制FGFRs和ERK磷酸化(见图7)，在与FGF-2共同诱导时二者都能抑制FGFRs和ERK的激活，且msFGFR2c较wsFGFR2c的抑制作用更强。
实施例9等温滴定量热法(iTC)检测FGFR2c胞外段146-356与EGFR的相互作用
本实验描述由实例1所描述的方法制备的msFGFR2c与EGFR结合的实验，所得实验结果如图8所示。
用等温滴定法(isothermal Titration Calorimetry，iTC)缓冲液(25mM HEPES，0.15mMNaCl， 5％甘油，pH＝7.5)分别透析msFGFR2c和自备的EGFR胞外段。透析完成的蛋白4℃、18000rpm离心30min后用于ITC实验。取200ul的EGFR加入到样品池中，样品浓度分别为20μmol/L。然后取150μmol/L的msFGFR2c40μL加入到上样针中用于滴定，反应条件为首次滴定0.4μL，其余每次滴定3μL，每2min滴定一次，总共滴定14次。
结果表明msFGFR2c能够与EGFR能够发生相互作用，具有良好的结合效果。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (11)

  1. 一种分离的蛋白质，其包含下述氨基酸序列：
    i).SEQ ID NO：1或2所述的氨基酸序列。
  2. 一种分离的蛋白质，其包含下述氨基酸序列，且具有抑制EGF信号、但不激活FGF信号的功能：
    ii).在SEQ ID NO：1或2所述的氨基酸序列中缺失、取代、***和/或添加1个或多个氨基酸而得的氨基酸序列；
    iii).与SEQ ID NO：1或2所述的氨基酸序列具有80％以上的氨基酸同源性的氨基酸序列；或者
    vi).由在严格条件下与编码SEQ ID NO：1或2所述的氨基酸序列的核酸的互补链杂交的核酸编码的氨基酸序列。
  3. 根据权利要求2所述的分离的蛋白质，其中，该分离的蛋白质来源于人。
  4. 编码权利要求1～3中任一项所述的分离的蛋白质的分离的核酸。
  5. 一种含有权利要求4所述核酸的载体。
  6. 一种含有权利要求5所述载体的宿主细胞。
  7. 根据权利要求6中所述的宿主细胞，其特征在于所述宿主细胞是CHO细胞、大肠杆菌细胞、昆虫细胞、酵母细胞中的任一种。
  8. 一种融合蛋白，其是权利要求1～3中任一项所述的分离的蛋白质与其他多肽的融合蛋白。
  9. 根据权利要求8所述的融合蛋白，其是权利要求1～3中任一项所述的分离的蛋白质与人免疫球蛋白表位标记序列或人免疫球蛋白Fc段的融合蛋白。
  10. 权利要求1～3中任一项所述的分离的蛋白质、权利要求4所述的核酸、权利要求5所述的载体、权利要求6或7所述的宿主细胞或权利要求8或9所述的融合蛋白在制备用于治疗恶性肿瘤的药物中的用途。
  11. 一种治疗恶性肿瘤的药物组合物，其包含权利要求1～3中任一项所述的分离的蛋白质、权利要求4所述的核酸、权利要求5所述的载体、权利要求6或7所述的宿主细胞或权利要求8或9所述的融合蛋白作为活性成分，以及药学上可接受的载体。
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