WO2007140667A1 - A ptd-vp3 fusion protein as anti-tumor medecine and preparing process - Google Patents

A ptd-vp3 fusion protein as anti-tumor medecine and preparing process Download PDF

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WO2007140667A1
WO2007140667A1 PCT/CN2006/003636 CN2006003636W WO2007140667A1 WO 2007140667 A1 WO2007140667 A1 WO 2007140667A1 CN 2006003636 W CN2006003636 W CN 2006003636W WO 2007140667 A1 WO2007140667 A1 WO 2007140667A1
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ptd4
protein
fusion protein
transduction domain
chicken anemia
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French (fr)
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Shen Qu
Jun Sun
Yiqiang Zong
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Huazhong University of Science and Technology
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Huazhong University of Science and Technology
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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present patent application relates to biotechnology, and more particularly to biotechnological drugs, and more particularly to biotechnological drugs for treating tumors.
  • Tumors are the main diseases causing death in the world.
  • the treatment methods for tumors include surgical treatment, radiotherapy and chemotherapy.
  • Surgical treatment has only a certain effect on early tumors, and is not effective for advanced tumors, radiotherapy and chemotherapy. High cost and great damage to human body. Finding new safe and effective drugs and methods for treating tumors is an important issue in the current medical field.
  • the VP3 protein also known as apoptin
  • CAV chicken anemia virus
  • VP3 (referred to as VP3 protein of chicken anemia virus, CAV VP3) can induce apoptosis of various tumor cells, and its induced apoptosis effect is non-p53-dependent [2] Not inhibited by anti-apoptotic factors Bel-2, Bel-xL [3] . More significantly, VP3 only induced apoptosis with tumorigenic phenotypic cells or transformed phenotype cells, whereas TP3 transgenic mice with normal apoptotic and non-cytotoxic effects on TH cells were able to grow normally [51 , these characteristics It indicates that VP3 is a promising anti-tumor preparation.
  • Protein transduction technology is a new macromolecular transfer technology in recent years, which utilizes some protei n transducting domain (PTD) (Chinese abbreviation: protein transduction domain). Proteins or peptides directly "transfer" therapeutic macromolecules into cells to exert biological effects [1Q] .
  • PTD protei n transducting domain
  • CPPs Cell l-Penetrati ng Peptides
  • ⁇ 11 peptide, 13 peptide
  • Antp 16 peptide
  • VP22 34 peptide
  • Transportan 28 peptide
  • MAP MAP
  • CPP can transport a wide range of substances, such as proteins, DNA, antibodies, imaging agents, toxins, nano drug particles, liposomes, etc. [1 ' ] .
  • the CPP delivery system is both a good tool for studying intracellular biological processes and a tool for biopharmaceutical delivery.
  • TAT trans-activator of transcription
  • the TAT protein (86 peptide) is a transcriptional activator protein.
  • Green [ 11] and Frankel [12] independently found that HIV-TAT protein has the ability to penetrate biofilm [ 12-13] ; The ability is mediated by a peptide between 47-57 (or 48-60) amino acid residues, and the fusion protein formed by the TAT polypeptide with other proteins can also penetrate the cell membrane and exert the biological functions of these proteins [ 14 _ 16] .
  • Ho et al. [17] used a chemical synthesis method to transform TAT, and obtained a series of TAT-PTD peptides by amino acid substitution.
  • the secondary structure of PTD4 is more stable and the transduction efficiency is higher, which can successfully transduce the target protein.
  • the rate is almost 100%, and the amount of protein introduced by a single cell is 33 times that of TAT.
  • whether the series of PTD can realize transmembrane transport of fusion protein through biosynthesis has not been reported yet. Summary of the invention
  • the object of the present invention is to provide a tumor transduction domain-chicken anemia virus protein 3 fusion protein (PTD-VP3 fusion protein, or PTD-CAV VP3 fusion protein), which has a good penetration biofilm effect. Moreover, it can induce tumor cell apoptosis, has greater lethality to tumor cells, does not damage normal cells, and does not have the introduction of foreign genes, especially the fusion protein drug can be applied by skin application. medicine. At the same time, the present invention also provides a method for preparing such a tumor-treating fusion protein.
  • PTD-VP3 fusion protein or PTD-CAV VP3 fusion protein
  • the technical scheme for realizing the present invention is: preparing a protein transduction domain-chicken anemia virus protein 3 fusion protein PTD-VP3, and directly introducing the VP3 protein into the cell by using the protein transduction domain PTD, and then specifically inducing apoptosis of the tumor cell by using VP3. Characteristics, thereby achieving the purpose of promoting tumor apoptosis and inhibiting tumor growth.
  • the PTD4-VP3 fusion protein was prepared, and the VP3 protein was directly introduced into the cell by PTD4, and then the VP3 was specifically used to induce the apoptosis of the tumor cell, thereby promoting tumor apoptosis and inhibiting tumor growth. purpose.
  • the protein transduction domain 4-chicken anemia virus protein 3 fusion protein provided by the invention has the amino acid sequence shown in sequence 6 or sequence 7 in the sequence listing; the expressed protein transduction domain 4-chicken anemia virus protein 3 fusion protein provided by the invention
  • the gene has the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing;
  • the method for preparing the protein transduction domain 4-chicken anemia virus protein 3 fusion protein provided by the present invention comprises the following steps: a. constructing prokaryotic expression containing the PTD4 sequence Vector pET28a- PTD4;
  • the method for constructing the prokaryotic expression vector pET28a-PTD4 containing the PTD4 sequence as described in a. above is:
  • the 11 amino acid sequence of the PTD4 polypeptide is designed according to the characteristics of the prokaryotic codon, and the two oligonucleotide base sequences encoding the PTD4 polypeptide are designed.
  • the two oligonucleotide base sequences encoding the PTD4 polypeptide have the sequence 2 in the sequence listing.
  • the nucleotide sequence shown, the two oligonucleotide fragments were synthesized, and the two oligonucleotide fragments were mixed in an equimolar manner at 95 ° C for 10 min, then left at room temperature for 1 h to form double-stranded DNA encoding PTD4.
  • the obtained DNA double strand encoding PTD4 was inserted into Nhe I of the prokaryotic expression vector pET28a.
  • the method for constructing the prokaryotic expression vector pET28a-PTD4-VP3 containing the PTD4-VP3 gene described in the above b. is: constructing the eukaryotic expression vector pcDNA-VP3 containing the VP3 gene, and having the restriction endonuclease at the 5' end
  • the two PCR primers of EcoR I and Xho I recognition sites 5'-AGGAATTCATGAACGCTCTCCAAG-3' and 5'-GCGTCGACTTACAGTCTTATACGCC-3' were subjected to PCR amplification reaction to amplify the VP3 gene of chicken anemia virus under the reaction condition of 94.
  • the purified recombinant plasmid pET28a-PTD4-VP3 was transformed into the expression strain Escherichia coli BL21 (DE3) PlysS in 5 ml, containing 0.05 mg/ml, and the purified recombinant plasmid pET28a-PTD4-VP3 was expressed in the above.
  • the purified recombinant plasmid pET28a-PTD4-VP3 was expressed in the above.
  • OmM was induced for 8 hours, sonicated, and the supernatant was collected by centrifugation to induce bacteria
  • a control identified by 12.5% SDS-PAGE
  • the supernatant was dissolved in the loading buffer (using the loading buffer indicated in the operating manual provided by the company using the purchased nickel affinity chromatography column)
  • the nickel affinity chromatography column was purified, and the eluted protein was identified by SDS-PAGE.
  • the eluate containing the target protein was combined, dialyzed, concentrated, filtered and sterilized, and the protein content was determined by BCA method, and stored at -80 °C.
  • the PTD4 synthesized by the present invention has a function of mediating the penetration of the fusion protein into the biofilm. It was confirmed by TUNEL method and DAPI staining that the PTD4-VP3 fusion protein provided by the present invention can induce tumor cell apoptosis.
  • the PTD4-VP3 fusion protein provided by the present invention has a good penetrating biofilm effect and can induce tumor cell apoptosis, and has great killing ability to tumor cells.
  • the protein transduction domain 4-chicken anemia fusion protein 3 (PTD4-VP3) provided by the invention can be used for preparing an anti-tumor drug, for example, for preparing an anti-liver cancer drug, and the preparation method comprises the PTD4-VP3 fusion protein as an active ingredient, plus
  • the pharmaceutically acceptable carrier and/or additive is prepared into an antitumor pharmaceutical preparation in a conventional manner.
  • the carrier may be PBS (phosphate buffer), glycerol or urea or the like.
  • the fusion protein provided by the present invention is used as an active ingredient, and a carrier for preparing a transdermal preparation, such as phosphate buffer, glycerin, water, petrolatum, polyethylene glycol, etc., can be prepared into a transdermal preparation for treating tumors. .
  • a carrier for preparing a transdermal preparation such as phosphate buffer, glycerin, water, petrolatum, polyethylene glycol, etc.
  • Figure 1 is a schematic diagram showing the construction of a recombinant plasmid of the present invention
  • Figure 6 shows the results of fluorescence microscopy of the transmembrane effect of the PTD4-GFP fusion protein
  • Figure 7 shows the results of optical microscopy of the transmembrane effect of the PTD4-GFP fusion protein
  • PTD4-VP3 fusion protein induces apoptosis of HepG 2 cells by laser confocal microscopy to observe FITC excitation results
  • Figure 9 PTD4-VP3 fusion protein induces apoptosis of HepG 2 cells by laser confocal microscopy to observe DAPI excitation results
  • Figure 10 PTD4- The results of FITC and DAPI co-excitation were observed by laser confocal microscopy of VP3 fusion protein induced apoptosis of HepG 2 cells.
  • FIG 11 PTD4-VP3 fusion protein induced apoptosis of HepG2 cells by laser confocal microscopy;
  • Figure 12 Human cervical cancer Hela cell tumor-bearing nude mice tumor growth curve;
  • PTD4 polypeptide consists of 11 amino acids, and its base sequence is designed according to the characteristics of prokaryotic codons as follows:
  • the prokaryotic expression vector pET- 28a (+) was purchased from Novagen.
  • the double strand obtained by the above method was inserted into Nhe I of pET28a, and DH5 ⁇ was transformed, and the recombinant plasmid pET28a-PDD4 was constructed by restriction enzyme digestion, PCR and sequencing. DNA sequencing was performed by Shanghai Boya Company.
  • the primers for PCR identification are:
  • the cycle parameters were 95 ⁇ denaturation 5 min, 94 °C lmin, 50 °C lmin, 72 °C lmin, cycle amplification 30 times, and finally 72 °C extension 10 min e
  • Ml Mraker, molecular weight standard, from large to small, 7000, 5500, 3500, 2000, 1000, 500bp A: pET28a-PTD4 after Nhe I digestion
  • M2 Mraker, molecular weight standard, 600, 500, 400, 300, 200, lOObp from large to small. From the results of Figure 2, the recombinant pET28a-PTD4 was successfully constructed.
  • the 5' ends of the two primers contain recognition sites for the restriction enzymes EcoR I and Xho I Sal I, respectively.
  • PCR amplification reaction conditions The cycle parameters were 94 ° C for 5 min, 94 ° C for 55 sec, 60 ° C for 50 sec, 72 ° C for 55 sec, cycle amplification for 30 times, and incubation at 72 ° C for 10 min.
  • the PCR product was identified by 1.5% agarose gel electrophoresis.
  • the construction method uses a conventional gene cloning method.
  • PCR amplification fragment VP3 and pET28a- PTD4 recombinant plasmids were digested with EcoR I and Sal I, respectively, and the target fragment was recovered, ligated and transformed into DH5 a, and the plasmid was amplified.
  • Figure 3 shows the results of identification of recombinant pET28a- PTD4- VP3, in which:
  • Ml Marker, molecular weight standard, from 7000, 5500, 3500, 2000, 1000, 500bp
  • M3 raker, molecular weight standard, from large to small, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, lOObp
  • pEGFP-Cl was purchased from CLONETECH.
  • the 5' ends of the two primers contain recognition sites for the restriction enzymes BamH I and EcoR I, respectively.
  • the cycle parameters were 94 ° C for 5 min, 94 ° C for 55 sec, 62 ° C for 55 sec, 72 ° C for lmin, cycle amplification for 30 times, and finally for 72 min at 72 ° C.
  • the PCR product was identified by the 1.5% agarose gel electrophoresis.
  • step (1) The construction of prokaryotic expression vector pET28a PTD4 is shown in step (1).
  • Figure 4 shows the results of identification of recombinant pET28a- PTD4-GFP, in which:
  • Ml Marker, molecular weight standard, from 7000, 5500, 3500, 2000, 1000, 500bp
  • the purified two recombinant plasmids pET28a-PTD4-VP3 and pET28a-PTD4-GFP were transformed into the expression strain Escherichia coli BL2KDE3) PlysS, respectively, and shaken in 5 ml of LB medium containing 0.05 mg/ml kanamycin at 37 °C. The 5% of the SDS- was added to the A leg. The sputum was added to the final concentration. The OMP was induced for 8 hours, and the supernatant was removed by ultrasound. The supernatant was collected by centrifugation. PAGE identification.
  • the supernatant was dissolved in a loading buffer and purified by a nickel affinity chromatography column, and the operation was carried out according to the requirements of the kit.
  • the eluted protein was identified by SDS-PAGE, and the eluate containing the protein of interest was combined, dialyzed, concentrated, filtered and sterilized, and the protein content was determined by BCA method, - 8 (TC storage.
  • M is the molecular weight standard of the protein, from large to small: 116. 0 kDa, 66. 2 kDa, 45. 0 kDa, 35. 0 kDa, 25. 0 kDa, 18. 4kDa, 14. 4kDa.
  • PTD4-GFP in PBS phosphate buffer: 0. 8% NaCl, 0.02% KC1, 0. 144% ⁇ 3 ⁇ 4 ⁇ 0 formulate 0. 024% ⁇ 3 ⁇ 4 ⁇ 0 medicine.
  • the human hepatoma cell line pG 2 was inoculated into the culture plate. After the cells were attached, the cells were incubated with the mol/L fusion protein PTD4-GFP. After 2 hours, the culture solution was aspirated, washed three times with PBS, and then observed under a fluorescence microscope.
  • Fig. 6 is a fluorescence microscope for the membrane transmembrane effect of PTD4-GFP fusion protein. The results of the observation; Fig. 7 shows the results of optical microscopic observation of the transmembrane effect test of the PTD4-GFP fusion protein.
  • FIG. 7 of the result indicating success PTD4- GFP fusion protein through the cell membrane into the cells in G 2 H printed, synthesized PTD4 described the present invention provides a fusion protein having the ability to mediate permeable membrane.
  • PTD4-VP3 in PBS phosphate buffer: 0. 8% NaCl, 0.02% KC1, 0. 144% ⁇ 3 ⁇ 4 ⁇ 0 formulate 0. 024% ⁇ 2 ⁇ 0, ) solution.
  • the experimental cells were purchased from human hepatocarcinoma cell line ⁇ G 2 to purchase CCTCC.
  • the TUNEL test kit was purchased from Roche. 2.
  • the treated coverslips were placed in a six-well plate, human hepatoma cells HepG 2 were seeded in a six-well plate at a suitable density, and after ligation of the cells, lnol/L fusion proteins PTD4-VP3 and PTD4-GFP were used.
  • - VP3 was incubated for 4-5 days, washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, apoptosis was detected by TUNEL (FITC staining) and counterstained with DAPI. The procedure was followed according to the instructions. Placed under a laser confocal microscope.
  • the PTD4-VP3 fusion protein provided by the present invention is used as an active ingredient, and PBS (phosphate buffer: 0.8% NaCl, 0.02% KC1, 0. 144% N3 ⁇ 4 HP0 4 , 0. 024% KH 2 P ( 3 ⁇ 4)
  • PBS phosphate buffer: 0.8% NaCl, 0.02% KC1, 0. 144% N3 ⁇ 4 HP0 4 , 0. 024% KH 2 P ( 3 ⁇ 4)
  • the solution is prepared into an antitumor drug preparation according to a conventional method.
  • the PTD4-VP3 fusion protein provided by the present invention is used as an active ingredient, and a PBS solution and 10% glycerol are added, and an antitumor drug preparation is prepared in a conventional manner.
  • the PTM-VP3 fusion protein provided by the present invention is used as an active ingredient, and a certain amount of urea (e.g., 4 mol/L) is added to prepare an antitumor drug preparation according to a conventional method.
  • urea e.g. 4 mol/L
  • the fusion protein concentrations of the above four drugs are generally in the range of 0.5-5.0 ⁇ 9 / ⁇ , and the dose is 4-12 g of fusion protein per g of nude mice.
  • Hela cells were cultured in RPMI 1640 medium, 10% calf serum, 37 ° C, 5% CO 2 culture. Receive The cultured cells were collected and injected into the roots of the hind limbs of nude mice at 2 X I0 6 / 0.1 mL / each.
  • nude mice After subcutaneous xenografts in nude mice were grown for 3 days (Hela), nude mice were randomly divided into experimental group and blank control group, with each group being thousands.
  • the blank control group was applied with PBS solution (or PBS glycerin solution) on the surface of the tumor
  • the experimental group was applied with the fusion protein PBS solution (or PBS glycerin solution) on the surface of the tumor; the application dose was controlled at 100 g/mouse as required.
  • the treatment was performed for 7 days, measured twice, and statistically analyzed for efficacy evaluation.
  • FIG. 12 The tumor growth curve of human cervical cancer Hela cell-bearing nude mice is shown in Figure 12.
  • Figure 13 shows the anti-tumor effect of PTD4-VP3 fusion protein on human cervical cancer Hela cell-bearing nude mice.
  • PTD4-VP3 fusion protein has the effect of inhibiting the tumor growth of human cervical cancer-bearing nude mice, and the fusion protein has the effect of treating cervical cancer.
  • mice Balb/C nude mice, male or female, 4-5 weeks old, weighing 15-20g. Purchased from the animal center of Hubei Provincial Epidemic Prevention Station;
  • SGC7901 cells were cultured in RPMI 1640 medium, 10% calf serum, 37 ° C, 5% C ⁇ 2 culture. The cultured cells were collected and injected into the roots of the hind limbs of nude mice at 2 X I0 6 / O.lmL.
  • PTD4-VP3 fusion protein was used to treat human gastric cancer.
  • SGC7901 cells were tumor-bearing in nude mice. The results are shown in the table below -
  • FIG. 14 The tumor growth curve of human gastric cancer SGC7901 cells in nude mice is shown in Figure 14.
  • Figure 15 shows the antitumor effect of PTD4-VP3 fusion protein on human gastric cancer SGC7901 cells in nude mice.
  • PTD4-VP3 fusion protein has the effect of inhibiting tumor growth in human gastric cancer-bearing nude mice, and the fusion protein has the effect of treating gastric cancer.
  • Example 12
  • HepG 2 cells were cultured in RPMM 640 medium, 10% calf serum, 37 ° C, 5% CO 2 culture. The cultured cells were collected and injected into the roots of the hind limbs of nude mice at 2 X I0 6 / 0.1 mL / each.
  • the tumor tumor block size of nude mice treated with PTD4-VP3 fusion protein in human hepatoma HepG 2 cells 7 days after inoculation is shown in the following table: Anti-tumor effect of white on human hepatocellular carcinoma HepG 2 cell-bearing nude mice.
  • the PTD4-VP3 fusion protein has the effect of inhibiting the tumor growth of human hepatocarcinoma-bearing nude mice, and the fusion protein has the effect of treating liver cancer.
  • the fusion protein formed by other protein transduction domains and CAV VP3 has the same therapeutic effect as the PTD4-VP3 fusion protein in treating tumors by dermal route.
  • the sequences of the nucleotides and amino acids involved in the patent application of the present invention are shown in the sequence listing, and the sequences in the table are:
  • Sequence 1 is a PTD4 coding strand DNA sequence
  • Sequence 2 is the amino acid sequence of PTD4;
  • Sequence 3 is the DNA sequence of chicken anemia virus vp3;
  • Sequence 4 is the amino acid sequence of chicken anemia virus V p3;
  • Sequence 5 is the DNA sequence of the PTD4-VP3 fusion protein
  • Sequence 6 is the amino acid sequence of the PTD4-VP3 fusion protein
  • Sequence 7 is the amino acid sequence of the PTD4-VP3 fusion protein digested by thrombin.
  • the references referred to in the patent application of the present invention are as follows:

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Description

一种抗肿瘤药物 PTD-VP3融合蛋白及其制备方法 技术领域
本发明专利申请涉及生物技术， 特别涉及生物技术药物， 尤其涉及用于治疗肿瘤的生物 技术药物。
背景技术 肿瘤是世界上致人死亡的主要疾病， 目前对肿瘤的治疗方法有手术治疗、 放疗和化疗 等措施， 手术治疗仅对早期肿瘤有一定疗效， 对晚期肿瘤则疗效不佳， 放疗和化疗费用高、 对人体损伤大， 寻找新的安全有效的治疗肿瘤的药物和方法， 是当前医药领域面临的重要课 题。 来源于鸡贫血病毒 ( chicken anemia virus, CAV)的 VP3蛋白 （亦称凋亡素， Apoptin ) 因其肿瘤特异性凋亡诱导效应而引起了人们的关注。体内外研究结果均表明， VP3 (本文中的 VP3均指鸡贫血病毒的 VP3蛋白，即 CAV VP3)能诱导多种肿瘤细胞凋亡 ， 其诱导的凋亡效 应为非 p53依赖性 [2]、 不受抗凋亡因子 Bel- 2、 Bel- xL的抑制 [3]。 更有意义的是， VP3仅诱 导具有致瘤表型细胞或转化表型细胞的凋亡， 而对 TH常细胞无凋亡效应、无细胞毒性 VP3 转基因小鼠能正常生长发育 [51， 这些特点表明 VP3是一种极有应用前景的抗肿瘤制剂，人们. 为利用 VP3治疗肿瘤进行了积极的探索。 目前， 直接导入外源性 vp3基因进行基因治疗仍是 人们采取的主要策略； 同时， 针对不同肿瘤尝试了不同策略以实现靶向性治疗 ί6—7]， 如我们 在国际上首次报道了通过受体介导的转移技术实现 νρ3基因体内肝细胞定向转移和表达，特 异性诱导肝癌细胞凋亡， 取得了良好的抑瘤效果[8— 9]。但是， 如何充分发挥 VP3 "肿瘤细胞特 异性诱导肿瘤细胞凋亡"的特性， 避免外源基因导入体内后可能出现的潜在风险， 如过度表 达等， 进一步扩展 VP3治疗肿瘤的应用范围， 仍是亟待解决的问题。 蛋白质转导技术 ( protein transduction technology ) 是近年来新兴的一种大分子转 移技术， 该技术利用一些具有蛋白质转导结构域（protei n transduct ion domain , PTD ) (中 文简称： 蛋白转导域） 的蛋白质或多肽， 直接将具有治疗作用的生物大分子 "送"入细胞发 挥生物学效应 [1Q]。 这种具有穿过细胞膜的能力的多肽称为细胞透膜肽 （Cel l- Penetrati ng Peptides , CPPs ) , 其长度一般不超过 30个氨基酸且富含碱性氨基酸， 如 ΤΑΤ ( 11肽、 13 肽） 、 Antp ( 16肽） 、 VP22 ( 34肽） 、 Transportan ( 28肽） 、 MAP ( 18肽） 。 CPP可传送 的物质范围很广泛， 如蛋白质、 DNA、 抗体、 显像试剂、 毒素、 纳米药物颗粒、 脂质体等[1']。 CPP传送系统既是一个很好的研究细胞内生物过程的工具， 也是一个在生物药物传送方面具
1
确 认 本 有潜在价值的研究对象。这一技术正成为肿瘤生物治疗的新思路——利用它可直接将治疗物 导入癌细胞， 使治疗作用更加可控 [1Q]。 目前相关研究多集中在 HIV-TAT ( trans-activator of transcription ) 多肽上。 TAT 蛋白 ( 86肽） 是一种转录激活蛋白， 1988年 Green[11]和 Frankel[12]各自独立发现 HIV-TAT 蛋白具有穿透生物膜的能力 [12— 13]； 随后发现这一透膜能力是由 47-57 (或 48-60 )氨基酸残 基之间的肽段所介导， 而且 TAT多肽与其他蛋白形成的融合蛋白也能透过细胞膜， 并能发挥 这些蛋白质的生物学功能 [14_16]。 Ho等人 [17]利用化学合成法对 TAT进行改造， 通过氨基酸替 换得到了一系列的 TAT- PTD多肽， 其中 PTD4的二级结构更稳定、 转导效率更高， 可使目标 蛋白转导成功率几乎达到 100%， 且单个细胞的蛋白质导入量是 TAT导入量的 33倍， 但该系 列 PTD能否通过生物合成而实现融合蛋白的跨膜运输目前尚未见报道。 发明内容
本发明的目的是提供一种治疗肿瘤的蛋白转导域 -鸡贫血病毒蛋白 3融合蛋白（PTD-VP3 融合蛋白， 或称 PTD-CAV VP3融合蛋白）， 使其具有良好的穿透生物膜效应且能诱导肿瘤细 胞凋亡、对肿瘤细胞具有较大杀伤力、 不损伤正常细胞、 不会有导入外源基因的情况发生等 特点， 特别是使这种融合蛋白药物能够实现经皮肤涂抹方式给药。 同时，本发明还提供这种 治疗肿瘤的融合蛋白的制备方法。
实现本发明的技术方案是： 制备蛋白转导域-鸡贫血病毒蛋白 3融合蛋白 PTD- VP3 , 利 用蛋白转导域 PTD直接将 VP3蛋白导入细胞，继而利用 VP3特异性地诱导肿瘤细胞凋亡的特 性， 从而实现促进肿瘤凋亡、 抑制肿瘤生长的目的。
以蛋白转导域 4为例， 制备 PTD4- VP3融合蛋白， 利用 PTD4直接将 VP3蛋白导入细胞， 继而利用 VP3特异性地诱导肿瘤细胞凋亡的特性， 从而实现促进肿瘤凋亡、 抑制肿瘤生长的 目的。本发明提供的蛋白转导域 4-鸡贫血病毒蛋白 3融合蛋白具有序列表中序列 6或序列 7 所示的氨基酸序列； 本发明提供的表达蛋白转导域 4-鸡贫血病毒蛋白 3融合蛋白的基因具 有序列表中序列 5所示的核苷酸序列； 本发明提供的制备蛋白转导域 4-鸡贫血病毒蛋白 3 融合蛋白的方法， 包括以下步骤- a.构建含 PTD4序列的原核表达载体 pET28a- PTD4;
b.构建含 PTD4- VP3基因的原核表达载体 pET28a- PTD4-VP3 ;
c. PTD4- VP3融合蛋白的表达及纯化。
上述 a.中所说的构建含 PTD4序列的原核表达载体 pET28a- PTD4 的方法是： 根据组成 PTD4多肽的 11个氨基酸序列， 按照原核生物密码子的特点设计编码 PTD4多肽的两条寡核 苷酸碱基序列， 该编码 PTD4多肽的两条寡核苷酸碱基序列具有序列表中序列 2所示的核苷 酸序列， 合成该两条寡核苷酸片段， 将该两条寡核苷酸片段等摩尔混合， 95°C 10min， 然后 室温放置 lh复性， 形成编码 PTD4的双链 DNA; 将获得的编码 PTD4的 DNA双链插入到原核 表达载体 pET28a的 Nhe I处。
前述 b.中所说的构建含 PTD4-VP3基因的原核表达载体 pET28a- PTD4- VP3的方法是： 构 建含 VP3基因的真核表达载体 pcDNA- VP3， 以 5'端分别含有限制性内切酶 EcoR I和 Xho I识 别 位 点 的 两 条 PCR 引 物 5'- AGGAATTCATGAACGCTCTCCAAG- 3' 和 5'- GCGTCGACTTACAGTCTTATACGCC- 3'进行 PCR扩增反应， 扩增鸡贫血病毒的 VP3基因， 反应条 件为循环参数为 94°C 5min, 94 °C 55sec 60 °C 50sec 72 °C 55sec , 循环扩增 30次， 72 °C保温 lOmin;将 PCR扩增片段 VP3和 pET28a- PTD4重组质粒分别经 EcoR I和 Sal I双酶切， 回收目的片段， 连接、 转化 DH5 a， 扩增提取质粒 pET28a_PTD4- VP3
前述 c.中所说的 PTD4- VP3 融合蛋白的表达及纯化的方法是： 将纯化后的重组质粒 pET28a-PTD4-VP3转化表达菌株大肠杆菌 BL21 (DE3) PlysS,在 5ml含 0. 05mg/ml卡那霉素的 LB培养基中 37 Ό震荡培养， 到 A =0. 4 0. 6时, 加入 IPTG至终浓度 1. OmM诱导 8小时， 超声破菌， 离心收集上清， 以未诱导菌作对照， 经 12. 5%SDS-PAGE鉴定； 将上清溶于上样缓 冲液中 （使用所购镍亲和层析柱的公司提供的操作手册上注明的上样缓冲液）， 经镍亲和层 析柱纯化， SDS - PAGE 鉴定洗脱蛋白， 合并含目的蛋白的洗脱液， 透析、 浓缩、 过滤除菌， BCA法测定蛋白含量， - 80°C保存。
通过蛋白转导实验证实， 本发明合成的 PTD4具有介导融合蛋白穿透生物膜的功能。 通 过 TUNEL法及 DAPI染色证实， 本发明提供的 PTD4- VP3融合蛋白能诱导肿瘤细胞凋亡。本发 明提供的 PTD4- VP3融合蛋白具有良好的穿透生物膜效应且能诱导肿瘤细胞凋亡， 对肿瘤细 胞具有极大的杀伤能力。
本发明提供的蛋白转导域 4-鸡贫血融合蛋白 3 ( PTD4-VP3 )可用来制备抗肿瘤药物， 如 用来制备抗肝癌药物， 其制备方法是以 PTD4- VP3融合蛋白为活性成分， 加上制药学上可接 受的载体和 /或添加剂， 按常规方法制备成抗肿瘤药物制剂。 所述的载体可以是 PBS (磷酸 盐缓冲液）、 甘油或尿素等。
以本发明提供的融合蛋白为活性成分， 加上制药学上制备透皮制剂所用载体， 如磷酸 盐缓冲液、 甘油、 水、 凡士林、 聚乙二醇等， 能制成治疗肿瘤的透皮制剂。
以下结合具体实施例和附图对本发明作进一步说明。 附图说明
图 1 本发明重组质粒构建示意图；
图 2 重组体 pET28a- PTD4鉴定结果； '
图 3 重组体 pET28a- PTD4-VP3鉴定结果；
图 4 重组体 pET28a- PTD4- GFP鉴定结果；
图 5 PTD4- GFP、 PTD4-VP3融合蛋白 PAGE结果；
图 6 PTD4-GFP融合蛋白的透膜效应实验的荧光显微镜观察的结果；
图 7 PTD4-GFP融合蛋白的透膜效应实验的光学显微镜观察的结果；
图 8 PTD4-VP3融合蛋白诱导 HepG2细胞凋亡的激光共聚焦显微镜观察 FITC激发结果； 图 9 PTD4-VP3融合蛋白诱导 HepG2细胞凋亡的激光共聚焦显微镜观察 DAPI激发结果； 图 10 PTD4-VP3融合蛋白诱导 HepG2细胞凋亡的激光共聚焦显微镜观察 FITC和 DAPI 共激发结果；
图 11 PTD4-VP3融合蛋白诱导 HepG2细胞凋亡的激光共聚焦显微镜光镜下结果； 图 12 人宫颈癌 Hela细胞荷瘤裸鼠的肿瘤生长曲线；
图 13 PTD4-VP3融合蛋白对人宫颈癌 Hela细胞荷瘤裸鼠的抑瘤效应；
图 14 人胃癌 SGC7901细胞荷瘤裸鼠的肿瘤生长曲线；
图 15 PTD4-VP3融合蛋白对人胃癌 SGC7901细胞荷瘤裸鼠的抑瘤效应；
图 16 人肝癌 HepG2细胞荷瘤裸鼠的肿瘤生长曲线；
图 17 PTD4-VP3融合蛋白对人肝癌 H印 G2细胞荷瘤裸鼠的抑瘤效应。 具体实施方式
实施例 1
构建含 PTD4序列的原核表达载体 pET28a- PTD4
1. 利用人工合成的寡核苷酸片段退火法制备 PTD4序列
( 1 )根据 Ho的报道 [171， PTD4多肽由 11个氨基酸组成， 按照原核生物密码子的特点自 主设计其碱基序列如下：
51： 5'-CTAGTTATGCCCGCGCGGCAGCACGACAAGCTCGAGCCC-3'
52： 5'-CTAGGGGCTCGAGCTTGTCGTGCTGCCGCGCGGGCATAA-3'
两条寡核苷酸片段由上海生物工程公司合成。
将两条寡核苷酸片段等摩尔混合， 95°C 10min， 然后室温放置 lh复性， 形成编码 PTD4 的双链 DNA。 2. 构建重组体 pET28a - PTD4
( 1 ) 原核表达载体 pET- 28a (+)购置 Novagen公司。
( 2 ) 构建方法釆用常规的基因克隆方法。
将上述方法获得的双链插入到 pET28a的 Nhe I处， 转化 DH5 α , 经酶切鉴定、 PCR、 测 序， 证明构建成 pET28a- PTD4重组质粒。 DNA序列测定由上海博亚公司完成。 PCR鉴定引物 为:
PI : GGCAGCACGACAAGCTCGAG
P2: AACCCCTCAAGACCCGTTTAGAG , 片段大小为： 298bp
PCR扩增反应条件
循环参数为 95Ό变性 5 min, 94°C lmin、 50 °C lmin、 72°C lmin , 循环扩增次数 30次， 最后 72°C延伸 10 min e
3. 重组质粒构建示意图见图 1。
4. 重组体 pET28a- PTD4鉴定结果见图 2, 图中
Ml : Mraker, 分子量标准， 从大到小依次为 7000, 5500, 3500, 2000, 1000， 500bp A: pET28a-PTD4经 Nhe I酶切后结果
B: 由步骤 1所获得的 PTD4片段
C: pET28a-PTD4 PCR 结果
M2: Mraker, 分子量标准， 从大到小依次为 600， 500， 400， 300， 200， lOObp 从图 2的结果可以判断重组体 pET28a- PTD4成功构建。
实施例 2
构建含 VP3基因的原核表达载体 pET28a- PTD4- VP3
1. 利用 PCR方法扩增鸡贫血病毒的 VP3基因
( 1 ) 构建含 VP3 基因的真核表达载体 pcDNA- VP3 , 构建方法参见： 王宇哲， 田俊， 屈 伸等， 鸡贫血病毒 VP3 基因的构建及体外凋亡诱导效应的研究， 同济医科大学学报， 2001， 30 (4)： 300-4。 1181
(2) PCR引物序列如下：
P5： 5'~AGGAATTCATGAACGCTCTCCAAG-3'
P6: 5'-GCGTCGACTTACAGTCTTATACGCC-3'
两条引物 5'端分别含有限制性内切酶 EcoR I和 Xho I Sal I的识别位点。
( 3 ) PCR扩增反应条件 循环参数为 94°C 5min， 94 °C 55sec、 60 °C 50sec、 72 °C 55sec, 循环扩增 30次， 72 °C保温 10min。 PCR产物经 1. 5%琼脂糖凝胶电泳鉴定无误。
2. 构建含 VP3基因的原核表达载体 pET28a- PTD4- VP3
( 1 ) 原核表达载体 pET28a-PTD4的构建见歩骤 （一）。
(2 ) 构建方法采用常规的基因克隆方法。
PCR扩增片段 VP3和 pET28a- PTD4重组质粒分别经经 EcoR I和 Sal I双酶切， 回收目的 片段， 连接、 转化 DH5 a， 扩增提取质粒。
3. 重组体鉴定结果见图 3。
图 3为重组体 pET28a- PTD4- VP3鉴定结果， 图中：
Ml : Marker, 分子量标准， 从大到小依次为 7000， 5500， 3500， 2000, 1000， 500bp
A: pET28a-PTD4-VP3经 EcoR I酶切
B: pET28a-PTD4-VP3经 EcoR I和 Sal I酶切
C: pcDNA-VP3的 PCR结果
M3 : raker, 分子量标准， 从大到小依次为 1500 , 1000， 900 , 800， 700， 600， 500， 400, 300, 200, lOObp
从图 3的结果可以判断重组体 pET28a- PTD4- VP3成功构建。
实施例 3
构建含 GFP基因的原核表达载体 pET28a- PTD4- GFP
1. 利用 PCR方法扩增绿色荧光蛋白的 GFP基因
( 1 ) pEGFP-Cl购自 CLONETECH公司。
(2 ) PCR引物序列如下：
P3： 5'- ACGGATCCATGGTGAGCAAGGGCG - 3'；
P4： 5' - GCGAATTCCTTGTACAGCTCGTCCATGC- 3'
两条引物 5'端分别含有限制性内切酶 BamH I和 EcoR I的识别位点。
( 3 ) PCR扩增反应条件
循环参数为 94°C 5min, 94 °C 55sec、 62 °C 55sec、 72 °C lmin, 循环扩增 30次， 最后 72°C保温 10min。 PCR产物经 1. 5%琼脂糖凝胶电泳鉴定条带大小正确。
2. 构建原核表达载体 pET28a- PTD4- GFP
( 1 ) 原核表达载体 pET28a PTD4的构建见步骤 （一）。
(2 ) 构建方法釆用常规的基因克隆方法。 PGR扩增片段 GFP和 pET28a-PTD 重组质粒分别经 BamH I和 EcoR I双酶切， 回收目的 片断， 连接、 转化 DH5 a，扩增提取质粒。
3. 重组体 pET28a- PTD4-GFP鉴定结果见图 4。
图 4为重组体 pET28a- PTD4- GFP鉴定结果， 图中：
Ml : Marker, 分子量标准， 从大到小依次为 7000， 5500， 3500， 2000, 1000, 500bp
A: 步骤 1所获得的 GFP片段
B: pET- 28a- PTD4- GFP经 BamH I和 EcoR I酶切
C: pET- 28a- PTD4- GFP经 BamH I酶切
从图 4的结果可以判断重组体 pET28a- PTD4- GFP成功构建。
实施例 4
PTD4 VP3、 PTD4-GFP融合蛋白的表达及纯化
1 . 原核表达菌株 E. col i BL2KDE3) PlysS 购置 Novagen 公司； 镍亲和层析柱购置 Novagen公司； IPTG购置 BBI公司； BCA蛋白测定试剂盒购置 Pi erce公司。
2. 融合蛋白表达过程
将纯化后的二种重组质粒 pET28a- PTD4- VP3和 pET28a- PTD4- GFP分别转化表达菌株大肠 杆菌 BL2KDE3) PlysS, 在 5ml 含 0. 05mg/ml 卡那霉素的 LB培养基中 37°C震荡培养， 到 A腿 =0. 4〜0. 6时, 加入 IPTG至终浓度 l. OmM诱导 8小时， 超声破菌， 离心收集上清， 以未诱 导菌作对照， 经 12. 5%SDS-PAGE鉴定。
3. 融合蛋白纯化过程
将上清溶于上样缓冲液中，经镍亲和层析柱纯化，操作歩骤按试剂盒要求进行。 SDS- PAGE 鉴定洗脱蛋白， 合并含目的蛋白的洗脱液， 透析、 浓缩、 过滤除菌， BCA法测定蛋白含量， - 8(TC保存。
4. 融合蛋白鉴定结果见图 5
图中电泳结果分别是：
1为纯化的 PTD4-VP3融合蛋白
2为 IPTG诱导 PTD4- VP3融合蛋白表达的细菌总蛋白；
3为未诱导表达的细菌总蛋白
M为蛋白质分子量标准， 从大到小依次为： 116. 0 kDa， 66. 2 kDa, 45. 0 kDa, 35. 0 kDa, 25. 0 kDa, 18. 4kDa， 14. 4kDa。
4为未诱导表达的细菌总蛋白 5为 IPTG诱导 PTD4-GFP融合蛋白表达的细菌总蛋白
6为纯化的 PTD4-GFP融合蛋白
图 5的电泳结果说明得到纯的 PTD4- VP3、 PTD4-GFP融合蛋白。
实施例 5
融合蛋白的体外透膜效应实验
1. 实验材料-
( 1 ) PTD4-GFP 的 PBS (磷酸盐缓冲液： 0. 8%NaCl， 0. 02%KC1, 0. 144%Ν¾ΗΡ0„ 0. 024%Κ¾Ρ0„) 溶液。
( 2 ) 实验细胞为人源肝癌细胞系 HepGs购置 CCTC (：。
2. 实验方法
将人源肝癌细胞 pG2接种于培养板中， 细胞贴壁后， 用 mol/L的融合蛋白 PTD4- GFP 孵育细胞， 2h后吸去培养液， PBS洗三次， 然后置于荧光显微镜下观察。
3. 实验结果
PTD4-GFP融合蛋白分别加入到体外培养的 HepG2细胞中， 2h后在细胞中可见明显的绿色 荧光， 见图 6和图 7， 图 6为 PTD4-GFP融合蛋白的透膜效应实验的荧光显微镜观察的结果； 图 7为 PTD4- GFP融合蛋白的透膜效应实验的光学显微镜观察的结果。 结合图 6、 图 7的结 果， 表明 PTD4- GFP融合蛋白成功透过细胞膜进入 H印 G2细胞中， 说明本发明提供的合成的 PTD4具有介导融合蛋白透膜的能力。
实施例 6
本发明融合蛋白诱导 HepG2细胞凋亡效应的实验
1. 实验材料-
( 1 ) PTD4-VP3 的 PBS (磷酸盐缓冲液： 0. 8%NaCl, 0. 02%KC1, 0. 144%Ν¾ΗΡ0„ 0. 024% Η2Ρ0, ) 溶液。
( 2 )实验细胞为人源肝癌细胞系 Η印 G2购置 CCTCC。 TUNEL检测试剂盒购置 Roche公司。 2. 实验方法
将处理过的盖玻片置于六孔板内， 人源肝癌细胞 HepG2以合适密度接种于六孔板中， 细 胞贴壁后，用 l nol/L的融合蛋白 PTD4- VP3及 PTD4- GFP- VP3孵育细胞 4-5天， PBS洗三次， 4%多聚甲醛固定 30min， TUNEL检测细胞凋亡 （FITC染色） 并用 DAPI复染， 操作过程按照 说明书进行。 置于激光共聚焦显微镜下观察。
3. 实验结果 融合蛋白 PTD4- VP3与 HepG2细胞孵育 4-5天， TUNEL检测细胞凋亡并用 DAPI复染， 激 光共聚焦显微镜下明显可见细胞核皱缩， 边集， 说明其融合蛋白能引起细胞凋亡， 见图 8、 9、 10、 1 1。 结合图 8、 9、 10、 1 1的结果， 说明 PTD4- VP3融合蛋白诱导 HepG2细胞发生凋亡 效应。
实施例 7
以本发明提供的 PTD4- VP3融合蛋白为活性成分， 加上 PBS (磷酸盐缓冲液： 0. 8%NaCl, 0. 02%KC1, 0. 144%N¾HP04, 0. 024%KH2P(¾ )溶液， 按常规方法制备成抗肿瘤药物制剂。
实施例 8
以本发明提供的 PTD4- VP3融合蛋白为活性成分， 加上 PBS溶液和 10%的甘油， 按常规 方法制备成抗肿瘤药物制剂。
实施例 9
以本发明提供的 PTM- VP3融合蛋白为活性成分， 加上一定量的尿素 （如 4mol/L)， 按 常规方法制备成抗肿瘤药物制剂。
实施例 10
本发明融合蛋白体内抑制宫颈癌生长效应的实验
1 . 实验材料
( 1 ) 动物： Balb/C裸小鼠， 雌雄不限， 4-5周龄， 体重 15-20g。 购自湖北省防疫站动 物中心
(2) 细胞株： 人宫颈癌细胞系 Hela细胞购置 CCTCC
(3) 治疗药物
( a ) PTD4-VP3 融合蛋白的 PBS (磷酸盐缓冲液： 0.8%NaCI， 0.02%KCI, 0.144%Na2HPO4, 0.024%KH2PO4) 溶液。
(b) PTD4-VP3融合蛋白的 PBS (同上) 甘油溶液 （含 10%甘油）。
(C) PTD4-VP3融合蛋白的水溶液。
(d) PTD4-VP3融合蛋白的 10%甘油水溶液。
以上 4种药物的融合蛋白浓度的范围一般为 0.5-5.0μ9/μΙ，使用剂量为每 g裸鼠 4-12 g 融合蛋白。
2. 实验方法
( 1 ) 荷瘤裸鼠动物模型的建立
Hela细胞均培养于 RPMI 1640培养基中， 10%的小牛血清， 37°C， 5% C02培养。 收 集培养细胞， 按 2 X I06个 /0.1mL/只， 注入裸鼠后肢根部皮下。
(2 ) 实验分组及处理
待裸鼠皮下移植瘤生长 3天（Hela)后， 将裸鼠随机分为实验组和空白对照组， 每组若 千只。 空白对照组在肿瘤表面涂抹 PBS溶液 （或 PBS甘油溶液）， 实验组在肿瘤表面涂抹 融合蛋白 PBS溶液 （或 PBS甘油溶液）； 涂抹剂量根据要求控制在 100 g/只。
(3) 肿瘤体积和瘤重的计算
治疗前测定肿瘤的长和宽 （为第 0天）， 以后每 3天测量 1次， 肿瘤体积按公式计算： 体积 =长 宽 2 X 0.52。 治疗 7天， 测量 2次， 统计学分析， 进行疗效评价。
3. 实验结果
接种 3天后 PTD4-VP3融合蛋白治疗人宫颈癌 Hela细胞荷瘤裸鼠的肿瘤瘤块大小结果 见下表:
Figure imgf000012_0001
人宫颈癌 Hela细胞荷瘤裸鼠的肿瘤生长曲线见图 12， 图 13为 PTD4-VP3融合蛋白对 人宫颈癌 Hela细胞荷瘤裸鼠的抑瘤效应。 结合图 12、 13说明 PTD4-VP3融合蛋白具有抑 制人宫颈癌荷瘤裸鼠的肿瘤生长效应， 该融合蛋白具有治疗宫颈癌的效应。 实施例 11
本发明融合蛋白体内抑制胃癌生长效应的实验
1 . 实验材料
( 1 ) 动物： Balb/C裸小鼠， 雌雄不限， 4-5周龄， 体重 15-20g。 购自湖北省防疫站动 物中心；
(2) 细胞株： 人胃癌细胞系 SGC7901购自 CCTCC;
(3) 治疗药物： 同实施例 10; .
2. 实验方法
( 1 ) 荷瘤裸鼠动物模型的建立
SGC7901细胞均培养于 RPMI 1640培养基中， 10%的小牛血清， 37°C， 5% C〇2培 养。 收集培养细胞， 按 2 X I06个 /O.lmL , 注入裸鼠后肢根部皮下。
(2) 实验分组及处理 待裸鼠皮下移植瘤生长 5天 （SGC7901 ) 后， 分组及处理方法同实施例 10。
(3) 肿瘤体积和瘤重的计算： 同实施例 10。
3. 实验结果
接种 5天后 PTD4-VP3融合蛋白治疗人胃癌 SGC7901细胞荷瘤裸鼠的肿瘤瘤块大 结果见下表-
Figure imgf000013_0002
人胃癌 SGC7901细胞荷瘤裸鼠的肿瘤生长曲线见图 14，图 15为 PTD4-VP3融合蛋白 对人胃癌 SGC7901细胞荷瘤裸鼠的抑瘤效应。 结合图 14、 15说明 PTD4-VP3融合蛋白具 有抑制人胃癌荷瘤裸鼠的肿瘤生长效应， 该融合蛋白具有治疗胃癌的效应。 实施例 12
本发明融合蛋白体内抑制肝癌生长效应的实验
1 . 实验材料
( 1 ) 动物： Balb/C裸小鼠， 雌雄不限， 4-5周龄， 体重 15-20g。 购自湖北省防疫站动 物中心
(2) 细胞株： 人肝癌细胞系 HepG2购置 CCTCC
(3) 治疗药物： 同实施例 10
2. 实验方法
( 1 ) 荷瘤裸鼠动物模型的建立
HepG2细胞均培养于 RPMM 640培养基中， 10%的小牛血清， 37°C， 5% C02培养。 收集培养细胞， 按 2 X I06个 /0.1 mL/只， 注入裸鼠后肢根部皮下。
(2) 实验分组及处理
待裸鼠皮下移植瘤生长 7天 （HepG2) 后， 分组及处理方法同实施例 10。
(3) 肿瘤体积和瘤重的计算： 同实施例 10
3. 实验结果
接种 7天后 PTD4-VP3融合蛋白治疗人肝癌 HepG2细胞荷瘤裸鼠的肿瘤瘤块大小结果 见下表：
Figure imgf000013_0001
Figure imgf000014_0001
白对 人肝癌 HepG2细胞荷瘤裸鼠的抑瘤效应。 结合图 16、 17说明 PTD4-VP3融合蛋白具有抑 制人肝癌荷瘤裸鼠的肿瘤生长效应， 该融合蛋白具有治疗肝癌的效应。
经实验，其它蛋白转导结构域与 CAV VP3形成的融合蛋白也具有 PTD4-VP3融合蛋白 经皮肤给药途径治疗肿瘤的同等疗效。 本发明专利申请涉及的核甘酸和氨基酸的序列见序列表， 表中序列依次为：
序列 1为 PTD4编码链 DNA序列；
序列 2为 PTD4的氨基酸序列；
序列 3为鸡贫血病毒 vp3的 DNA序列；
序列 4为鸡贫血病毒 Vp3的氨基酸序列；
序列 5为 PTD4-VP3融合蛋白的 DNA序列；
序列 6为 PTD4- VP3融合蛋白氨基酸序列；
序列 7为经凝血酶酶切后的 PTD4- VP3融合蛋白氨基酸序列。 本发明专利申请涉及的参考文献如下：
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Claims

权 利 要 求 书
1. 蛋白转导域-鸡贫血病毒蛋白 3融合蛋白 PTD-VP3。
2. 根据权利要求 1所述的蛋白转导域-鸡贫血病毒蛋白 3融合蛋白 PTD-VP3, 其特征 在于所述的蛋白转导域为蛋白转导域 4， 蛋白转导域 4-鸡贫血病毒蛋白 3 融合蛋白 PTD4-VP3具有序列表中序列 6或序列 7所述的氨基酸序列。
3. 表达蛋白转导域 4-鸡贫血病毒蛋白 3融合蛋白 PTD4-VP3的基因， 具有序列表中序 列 5所述的核苷酸序列。
4. 含有表达权利要求 1或权利要求 2所述融合蛋白的基因的表达载体。
5.含有表达权利要求 2所述的蛋白转导域 4-鸡贫血病毒蛋白 3融合蛋白 PTD4-VP3的 基因的表达载体 pET28a-PTD4-VP3。
6. 权利要求 1或 2所述的融合蛋白在制备抗肿瘤药物中的应用。
7. 一种治疗肿瘤的药物制剂， 其特征在于， 该药物制剂以权利要求 1或 2所述的融合 蛋白为活性成分。
8. 一种治疗肿瘤的透皮制剂， 其特征在于， 该透皮制剂以权利要求 1或 2所述的融合 蛋白为活性成分加上制药学上制备透皮制剂所用载体。
9. 根据权利要求 8所述的治疗肿瘤的透皮制剂， 其特征在于， 所述的载体选自磷酸盐 缓冲液、 甘油、 水、 凡士林和聚乙二醇。
10. 权利要求 2所述的蛋白转导域 4-鸡贫血病毒蛋白 3融合蛋白 PTD4-VP3的制备方 法， 包括以下步骤：
a.构建含 PTD4序列的原核表达载体 pET28a-PTD4;
b.构建含 PTD4-VP3基因的原核表达载体 pET28a-PTD4-VP3;
c. PTD4-VP3融合蛋白的表达及纯化。
11 . 根据权利要求 10所述的制备蛋白转导域 4-鸡贫血病毒蛋白 3融合蛋白的方法， 其 特征在于， 所说的构建含 PTD4序列的原核表达载体 pET28a-PTD4 的方法是： 根据组成 PTD4多肽的 11个氨基酸序列， 按照原核生物密码子的特点设计编码 PTD4多肽的两条寡 核苷酸碱基序列，合成该两条寡核苷酸片段，将该两条寡核苷酸片段等摩尔混合， 95°C 10min, 然后室温放置 lh复性， 形成编码 PTD4的双链 DNA, 将获得的编码 PTD4的 DNA双链插 入到原核表达载体 pET28a的 Nhe I处。
12. 根据权利要求 1 1所述的制备蛋白转导域 4鸡贫血病毒蛋白 3融合蛋白的方法， 其 特征在于所说的编码 PTD4多肽的两条寡核苷酸碱基序列具有序列表中序列 1所示的核苷酸 序列。
13. 根据权利要求 10所述的制备蛋白转导域 4-鸡贫血病毒蛋白 3融合蛋白的方法， 其 特征在于所说的构建含 PTD4-VP3基因的原核表达载体 pET28a-PTD4-VP3的方法是： 构 建含 VP3基因的真核表达载体 pcDNA-VP3， 以 5'端分别含有限制性内切酶 EcoR I和 Xho I 识 别 位 点 的 两 条 PCR 引 物 5'-AGGAATTCATGAACGCTCTCCAAG-3' 和 5'-GCGTCGACTTACAGTCTTATACGCC-3'进行 PCR扩增反应， 扩增鸡贫血病毒的 VP3 基因， 反应条件为循环参数为 94°C 5min， 94 °C 55sec、 60 °C 50sec、 72 °C 55sec, 循环 扩增 30次， 72°C保温 10min; 将 PCR扩增片段 VP3和 pET28a-PTD4重组质粒分别经 EcoR I和 Sai l双酶切， 回收目的片段， 连接、 转化 DH5a， 扩增提取质粒 pET28a-PTD4- VP3。
14. 根据权利要求 10所述的制备蛋白转导域 4-鸡贫血病毒蛋白 3融合蛋白的方法， 其 特征在于所说的 PTD4-VP3 融合蛋白的表达及纯化的方法是： 将纯化后的重组质粒 pET28a-PTD4-VP3转化表达菌株大肠杆菌 BL21 (DE3)PlysS，在 5ml含 0.05mg/ml卡那霉 素的 LB培养基中 37°C震荡培养， 到 A6(X)=0.4~0.6时， 加入 IPTG至终浓度 1.0mM诱导 8 小时， 超声破菌， 离心收集上清， 以未诱导菌作对照， 经 12.5%SDS-PAGE鉴定； 将上清 溶于上样缓冲液中， 经镍亲和层析柱纯化， SDS-PAGE 鉴定洗脱蛋白， 合并含目的蛋白的 洗脱液， 透析、 浓缩、 过滤除菌， BCA法测定蛋白含量， -8CTC保存。
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