WO2006089455A1 - Protéine de fusion antitumorale de ciblage comprenant la protéine adénovirale e4orf4 - Google Patents

Protéine de fusion antitumorale de ciblage comprenant la protéine adénovirale e4orf4 Download PDF

Info

Publication number
WO2006089455A1
WO2006089455A1 PCT/CN2005/000211 CN2005000211W WO2006089455A1 WO 2006089455 A1 WO2006089455 A1 WO 2006089455A1 CN 2005000211 W CN2005000211 W CN 2005000211W WO 2006089455 A1 WO2006089455 A1 WO 2006089455A1
Authority
WO
WIPO (PCT)
Prior art keywords
e4orf4
egf
fusion protein
protein
cells
Prior art date
Application number
PCT/CN2005/000211
Other languages
English (en)
Chinese (zh)
Inventor
Hong Chen
Bingren Huang
Xin Wang
Xiaoli Ma
Original Assignee
Institute Of Basic Medical Sciences, Chinese Academy Of Medical Sciences
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute Of Basic Medical Sciences, Chinese Academy Of Medical Sciences filed Critical Institute Of Basic Medical Sciences, Chinese Academy Of Medical Sciences
Priority to PCT/CN2005/000211 priority Critical patent/WO2006089455A1/fr
Publication of WO2006089455A1 publication Critical patent/WO2006089455A1/fr

Links

Classifications

    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Targeted anti-tumor fusion protein containing adenovirus E4orf4 protein is Targeted anti-tumor fusion protein containing adenovirus E4orf4 protein
  • the present invention relates to a recombinant human epidermal growth factor-adenovirus early transcribed region 4 fourth open reading frame protein product (E4orf4) fusion protein, a fusion gene encoding the fusion protein, an expression vector containing the fusion gene, and the use of methyl nutrition
  • E4orf4 fourth open reading frame protein product
  • the yeast is a method for secretory expression of such a recombinant fusion protein, a pharmaceutical composition containing the same, and a therapeutic use thereof.
  • the early transcribed region 4 (E4) transcription unit of the human adenovirus gene contains at least 7 open reading frames, and the respective products have no obvious similarity.
  • the product encoded by the fourth open reading frame 4 (E4orf4) is a 14kDa small molecule protein consisting of 114 amino acids, and its amino acid sequence has no homology with any known protein.
  • the E4orf4 protein has a highly conserved sequence, RXKRRXRRRR, which also has a proline-rich sequence at the amino terminus, potentially providing a potential Src homology region 3 (SH3) binding site. Deletion of any small portion of an amino acid in a polypeptide chain, including both the amino terminus and the carboxy terminus region, results in a non-functional and often unstable mutant.
  • the E4orf4 gene is transcribed from the E4 promoter to produce i RNA. Although the transcription was stopped in the late E4 promoter, the E4orf4 protein in the cells remained at a stable level. This indicates that the E4orf4 protein is highly stable in cells (Branton PE and Roopc and DE. Oncogene, 2001; (20): 7855-7865; Marcellus RC, Chan H, Paquette D et al. J Virol, 2000; 74 (17 ): 7869-7877).
  • E1A adenovirus early region 1A
  • E1B Early zone IB
  • E4orf4 protein has high cytotoxicity, induces apoptosis independent of p53, and pcDNA plasmid expressing E4orf4 can induce tumor cells. With a mortality rate of 80-90%, it is the only E4 product that kills cells by itself.
  • the adenovirus E4orf4 protein is a multifunctional viral regulatory protein that specifically induces apoptosis in transformed cells in vitro, but has no effect on normal cells; apoptosis induced by E4orf4 protein is p53-independent Its role in inducing apoptosis requires interaction with protein phosphatase 2A; E4orf4 regulates the activity of Src kinase and triggers cytoplasmic expression of apoptosis (Maecell s RC, Tendorn JG, Wu T et al. J Virol. 1996, 70:6207-6215; Marcellus RC, Lavoie JN, Boivin D et al. J. Virol. 1998, 72: 7144-7153; Shtricheman R, Sharf R, Barr H et al. Proc. Natl. Acad Sci. USA. 1999, 96: 10080-10085).
  • EGF receptors are overexpressed in most tumor cells such as breast, lung, brain, bladder, kidney, prostate, etc., and their cell lines are often inhibited by EGF after growth or transplantation in vitro.
  • the results showed that chemically linked RNase 1 and recombinant EGF form a complex EGF-RNase 1, which has a dose-dependent cytotoxic effect on breast cancer and squamous cell carcinoma with high expression of EGF receptor, ie, the more EGF receptor is expressed The higher the cell line, the more obvious the cytotoxic effect.
  • CAR-EGF fusion protein (CAR, Coxsackie virus and adenovirus receptor) using a baculovirus expression system, and efficiently transported adenoviral vectors through the targeting of EGF binding to EGF receptors.
  • Increased reporter gene release into tumor cells Yang Yu'an, Huang Bingren, Cai Liangbiao, et al. Journal of Chinese Academy of Medical Sciences. 1996, 18(3): 171; Ueda M, Psarras K, Ji, H et al. Breast Cancer 1997, 4(4): 253-255; Dmitriev I, Kashentseva E, Rogers BE et al. J. Virol. 2000, 74(15): 6875-6884).
  • Adenovirus is a promising gene therapy vector.
  • gene therapy for adenovirus provides many benefits, the technology is not yet fully mature, and there is a certain risk in the safety of treatment. Therefore, further exploration of methods and drugs for treatment with adenovirus remains a hot spot in the field of cancer therapy. Summary of invention
  • the present invention relates to a recombinant human epidermal growth factor-adenovirus E4orf4 fusion protein, a pharmaceutical composition containing the same, and its use for targeted tumor therapy.
  • the fusion gene encoding the fusion protein of the present invention has the sequence of SEQ ID NO: 1, and the amino acid sequence of the fusion protein is as set forth in SEQ ID NO: 2.
  • Another aspect of the invention relates to a fusion gene encoding the fusion protein, an expression vector containing the fusion gene, and an engineered strain.
  • a genetically engineered strain according to the present invention named Pichia pastoris GS115-3 EGF-E4orf4, deposited on July 15, 2002 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee, The number is 0771. The deposit was transferred to the international deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Patent Procedure on February 22, 2005.
  • Figure 1 Overlapping PCR to construct the ⁇ -factor leader peptide-EGF-E4orf4 gene fragment
  • Figure 2 Recombinant plasmid pUC-EGF-E4orf4 digestion and identification electrophoresis map
  • Figure 3 Recombinant plasmid pUC-EGF-E4orf4 site-directed mutagenesis
  • Figure 4 Physical map of plasmid pA0815, pA0815 consists of 7709 nucleotides, wherein base 1 940 is a 5 ⁇ 1 promoter fragment, bases 943-948 are EcoRJ sites, and bases 950-1277 are 3 ⁇ 1 transcription terminators ( TT), base 4199-1665 is HISORF, bases 4554-5310 are 3 ⁇ 1 fragments, bases 6394-5740 are starting with ColEl, and bases 7399-6539 are ampicillin resistance genes;
  • Figure 5 Construction of recombinant plasmid pAO-EGF-E4orf4;
  • FIG. 6 Electrophoresis pattern of the recombinant plasmid pAO-EGF-E4orf4;
  • Figure 7 Construction of recombinant plasmid pAO-3EGF-E4orf4;
  • FIG. 8 Electrophoresis pattern of the recombinant plasmid pAO-3EGF-E4orf4;
  • FIG. 10 SDS-PAGE electrophoresis pattern of EGF-E4orf4 secreted by GS115-3EGF-E4orf4;
  • Figure 11 Western Blot analysis of EG115-3EGF-E4orf4 secreting EGF-E4orf4.
  • Figure 12 Results of recombinant EGF-E4orf4 fusion protein purified by cation exchange chromatography
  • Figure 13 MTT colorimetric assay for BT325 cell viability
  • Figure 14 MTT colorimetric assay for MDA-MB-231 cell viability
  • FIG. 15 MDA-MB-231 cells were cultured for 72 hours, and apoptosis was analyzed by flow cytometry;
  • FIG. 16 MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (10 ⁇ ⁇ /3 ⁇ 10 5 cells) for 72 hours, and apoptosis was analyzed by flow cytometry;
  • FIG. 17 MDA-MB-231 cells were co-cultured with the fusion protein EGF_E4orf4 (15 ⁇ ⁇ /3 ⁇ 10 5 cells) for 72 hours, and apoptosis was analyzed by flow cytometry;
  • Figure 18 MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (20 ⁇ ⁇ /3 ⁇ 10 5 cells) for 72 hours, and apoptosis was analyzed by flow cytometry;
  • FIG. 19 MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (25 ⁇ ⁇ /3 ⁇ 10 5 cells) for 72 hours, and apoptosis was analyzed by flow cytometry. Detailed description of the invention
  • a recombinant human epidermal growth factor (EGF)-adenovirus E4orf4 fusion protein comprising human epidermal growth factor, a linker, and an adenovirus E4orf4 protein from N-terminal to C-terminal is provided.
  • the amino acid sequence of the fusion protein is set forth in SEQ ID NO: 2.
  • the present invention provides a fusion gene encoding the fusion protein of the present invention.
  • the artificial fusion gene encoding the 51 amino acid EGF gene fragment is used in the fusion gene of the present invention, and the gene fragment is described in Chinese Patent Application No. 97115284.5.
  • the E4orf4 protein gene sequence of the adenovirus in the fusion gene of the present invention is preferably a modified E4orf4 protein gene sequence encoding adenovirus A5, which is a site-directed mutation that mutates the 228th base from T to G, thereby The restriction endonuclease Bglll cleavage site was eliminated, but the amino acid sequence was not altered to facilitate the construction of the yeast multiple copy tandem expression vector.
  • the linker between the two gene fragments of EGF and E4orf4 is preferably a gene sequence encoding 11 amino acids consisting of glycine and serine, so that it can form an arm to ensure that EGF and E4orf4 proteins can not interfere with each other, and each forms an independent Advanced structure, exercising normal biological activity.
  • the gene sequence encoding the EGF-E4orf4 fusion protein of the present invention is represented by SEQ ID NO: 1.
  • an expression vector comprising the fusion gene of the present invention and an engineered bacteria containing the expression vector.
  • the expression vector of the present invention is An expression vector for secreted expression of an integrated single-copy or triple-copy EGF-E4orf4 fusion protein by fusing fusion of a DNA fragment encoding a yeast alpha-factor leader peptide with a gene sequence of an EGF-E4orf4 fusion protein, and The ⁇ -factor leader peptide was added to the Kozak sequence and cloned downstream of the alcohol oxidase promoter (AOX1).
  • AOX1 alcohol oxidase promoter
  • the engineered bacteria of the present invention is a methylotrophic yeast (also known as Pichia pastoris) comprising the expression vector of the present invention, particularly Pichia pastoris GS115-3 EGF-E4orf4, which was in 2002. Deposited on July 15th at the General Microbiology Center of China Microbial Culture Collection Management Committee
  • the strain is an integrated Mut + genotype strain (GS115-3 EGF-E4orf4) screened by transforming the Pichia pastoris strain GS115 into a 3-copy vector. After high-density culture and induced expression, the strain can secrete EGF- E4orf4 fusion protein. The molecular weight of the secreted EGF-E4orf4 fusion protein was consistent with the predicted. Western Blot analysis was performed using rabbit anti-human EGF polyclonal antibody and rabbit anti-adenovirus E4orf4 protein antiserum, respectively, and it was confirmed that the specific expressed protein band was positively reacted with both antibodies, and it was a fusion protein composed of EGF and E4orf4. . The expressed protein is separated and purified by column chromatography to have a purity of 90% or higher.
  • the present invention has two terminators TAA and TGA linked to the coding sequence of the 176th amino acid of the EGF-E4orf4 fusion protein. This design facilitates the isolation and purification of the gene expression product and the termination of gene transcription. The addition of the Kozak sequence provides conditions for efficient expression of the EGF-E4orf4 fusion protein.
  • the fusion protein of the invention has tumor targeting property and can specifically kill tumor cells, especially tumors deficient in p53 gene, and the targeting is to utilize the high expression of EGF receptor on the surface of most tumor cells, and the fusion protein contains EGF.
  • E4orf4 protein can be introduced into tumor cells by binding EGF to EGF receptor.
  • E4orf4 is a highly cytotoxic protein in adenovirus, which can induce tumor cell-independent p53. Death, thereby achieving targeted treatment of tumors.
  • the activity of the fusion proteins of the invention is verified as described in the Examples section.
  • compositions comprising a pharmaceutically effective amount of an EGF-E4orf4 fusion protein as an active ingredient.
  • Such compositions contain a pharmaceutically acceptable carrier, adjuvant, diluent or filler.
  • the pharmaceutical composition can be administered by injection or by topical administration to a tumor, in order to be therapeutically suitable for clinical use in a clinical setting.
  • Example 1 Overlapping PCR construction of ⁇ -factor leader peptide- EGF-E4orf4 gene fragment
  • PCR primers were used:
  • Primer 1 contains an EcoRI cleavage site and a Kozak sequence, and is complementary to the 5' end of the ⁇ -factor leader peptide; ligate 2 is complementary to the 3' end of EGF and incorporates an amino acid partially encoded by glycine and serine. Sequence of arms; primer 3 and 5, end of E4orf4 Supplement, and added a sequence encoding an amino acid arm consisting of glycine and serine, this sequence is complementary to primer 3; primer 4 is complementary to the 3' end of E4orf4, and two terminators and an EcoRI cleavage site are added.
  • plasmid pYA41EGF as a template (Huang Bingren, Zhang Wei, Chi Lai Shun, et al. Journal of Chinese Academy of Medical Sciences 1989, 11 (5 ): 331 -337), using primer 1 and primer 2 to obtain a DNA fragment containing an EcoRI recognition site. , Kozak sequence, ⁇ -factor leader peptide, EGF and 8 amino acid arms consisting of glycine and serine.
  • primer 3 and primer 4 contained 8 amino acid arms consisting of glycine and serine, E4orf4 and E coRI recognition sites.
  • the first annealing is performed to make the five amino acid bases of the two amino acid arms complementary, and then the primers 1 and the primers 4 are used for amplification, thereby obtaining the EcoRI recognition sites at both ends and containing the complete ⁇ -factor leader peptide-EGF-E4orf4 gene fragment.
  • the gene fragment was digested with EcoRI and ligated into the pUC18 plasmid vector, and the resulting recombinant clone was pUC-EGF-E4orf4, as shown in FIG.
  • the sequencing results confirmed that it was completely consistent with the design.
  • Example 2 Enzyme digestion identification of recombinant plasmid pUC-EGF-E4orf4
  • Fig. 2 The restriction enzyme digestion of the plasmid is shown in Fig. 2.
  • Lane 1 ⁇ / ⁇ , the fragments are: 23130 bp, 9414 bp, 6557 bp, 4361 bp, 2322 bp, 2021 bp, 564 bp.
  • Lane 2 Recombinant plasmid pUC-EGF-E4orf4.
  • Lane 3 pUC-EGF-E4orf4/EcoRI 2.69 kb (vector), 800 bp (insert).
  • Lane 4 DNA Marker DL2000, the fragments from large to small are: 2000 bp, 1000 bp, 750 bp, 500 bp, 250 bp, 100 bp.
  • Example 3 Site-directed mutagenesis of recombinant plasmid pUC-EGF-E4orf4
  • the exogenous gene fragments cannot contain the tangent points of Bglll and BamHI.
  • the E4orf4 gene fragment contains a Bglll recognition site, which is removed by site-directed mutagenesis, but does not alter the amino acid sequence.
  • the site-directed mutagenesis was performed using Stratagene's kit (QuickCliangeTM Site-Directed Mutagenesis Kit). The strategy is shown in Figure 3.
  • the primer sequences for site-directed mutagenesis are as follows:
  • the plasmid pUC-EGF-E4orf4 obtained in Example 1 was used as a template, and after PCR amplification of site-directed mutagenesis, Escherichia coli DH5a was transformed, and the transformant was subjected to DNA sequence. Analysis confirmed that the 228th base of the E4orf4 gene changed from T to G.
  • the pUC-EGF-E4orf4 plasmid was digested with EcoRI to obtain a gene fragment encoding the a-factor leader peptide-EGF_E4orf4, and ligated with the EcoRI-linearized yeast expression vector pA0815 (purchased from Iiivitrogen) to obtain the recombinant plasmid pAO-EGF. -E4orf4 ( Figure 5).
  • Example 4 Restriction enzyme digestion of recombinant plasmid pAO-EGF-E4orf4
  • the pA0815 vector AOX1 promoter was the EcoRI cloning site at the 944 bp downstream. After the recombinant plasmid pAO-EGF-E4orf4 was digested with EcoRI, an insert of about 800 bp was observed. The recombinant plasmid pAO-EGF-E4orf4 was screened by restriction enzyme digestion to identify the forward clone. There is a Pstl tangent point at the 24th base of the a-factor leader peptide, and a Pstl cut at the 6858th base of the pA0815 vector.
  • Fig. 6 The electrophoresis results of the restriction enzyme digestion of the plasmid are shown in Fig. 6.
  • Lane 1 DNA molecular weight marker DL2000, the fragments are from 2000 bp to 100 bp, 750 bp, 500 bp, 250 bp, and 100 bp.
  • Lane 2 pAO-EGF-E4orf4/EcoRI, 7.7 kb (vector), 800 bp (insert).
  • Lane 3 pAO-EGF-E4orf4/PstI, 6.7 kb, 1.8 kb.
  • Lane 4 pAO-EGF-E4orf4.
  • Lane 5 The DNA/HindllL fragments were: 23130 bp, 9414 bp, 6557 bp, 4361 bp, 2322 bp, 202 lbp, 564 bp in descending order.
  • Example 5 Construction of a three-copy expression vector pAO-3EGF-E4orf4
  • the pAO-EGF-E4orf4 obtained in Example 3 was excised with Bglll and BamHI to contain the AOX1 promoter, the ⁇ -factor leader peptide-EGF-E4orf4 coding sequence and the AOX1 terminator fragment, so that Bglll and Bglll were used in vitro.
  • BamHI was digested. At this time, only the dimer of Bglll and BamHI was formed in the reaction system, that is, the fragments of Bglll and BamHI at both ends of the fragment could not be cleaved again.
  • the reaction was ligated into the BamHI site of alkaline phosphatase-treated pAO-EGF-E4orf4, and transformed into DH5a receptor bacteria.
  • Three-copy EGF-E4orf4 expression unit containing the same direction was obtained after recombinant DNA digestion and identification.
  • the plasmid construction map is shown in Figure 7.
  • the plasmid restriction map is shown in Figure 8.
  • Lane 1 DNA/Hindlll, the fragments from big to small are: 23130 bp, 9414 bp, 6557 bp. 4361 bp, 2322 bp, 202 lbp, 564 bp.
  • Lane 2 pA0815/BglII+BamHI, the fragments from large to small are: 4028 bp, 2405 bp, 1279 bp.
  • Lane 3 pAO-EGF-E4orf4/ Bglll+BamHI, the fragments from large to small are: 4028 bp, 2405 bp, 2079 bp.
  • Lane 4 pAO-3EGF-E4orf4/ Bglll+BamHI, the fragments from large to small are: 6237 bp, 4028 bp, 2405 bp.
  • Example 6 Preparation, transformation and screening of host bacteria
  • Methylotrophic yeast i.e., Pichiapastoris, Pichia pastoris
  • Methylotrophic yeast has many advantages: (1) Contains the unique AOX1 (alcohol oxidase) promoter, which can be tightly regulated by methanol. (2) There is a complete fermentation method, which can be continuously cultured at a high density, and the protein expression yield is high. (3) It is highly viable, easy to handle, and can be grown in inexpensive non-selective media. To this end, the multicopy EGF-E4orf4 expression vector pAO-3EGF-E4orf4 obtained in Example 5, which was obtained by the present invention, was transformed, screened and expressed as follows.
  • 10X YNB 134 g of yeast base containing ammonium sulfate but no amino acid was dissolved in 1000 ml of water, sterile filtered and used.
  • 10X GY (10% glycerol): 100ml glycerol is dissolved in 900ml water, autoclaved, and stored at room temperature for use.
  • 1M phosphate buffer, pH 6.0 mixed 132ml 1M K 2 HP04, 868ml 1M
  • BMG and BMMY lOOmM phosphate buffer pH 6.0, 1.34% YNB, 1% glycerol or 0.5% methanol, 0.00004% biotin, BMMY also contains 1% yeast extract, 2% peptone.
  • YPD medium 1% yeast extract, 2% peptone, 2% glucose.
  • MD and MM 1.34% YNB, 0.00004% biotin, 2% glucose or 0.5% methanol.
  • Preparation of medium amount of plasmid DNA 50 ml of LB medium containing 100 ⁇ ⁇ ampicillin/ml was inoculated with the strain containing the expression vector, and cultured at 37 ° C overnight, and the cells were collected by centrifugation, and then the plasmid purification reagent of QIAGEN was used. The plasmid was extracted and purified, and 10 g of pAO-3EGF-E4orf4 was completely digested with Bglll, extracted with phenol-chloroform-isoamyl alcohol, ethanol precipitated, and lyophilized for use.
  • Example 7 pAO-3EGF-E4orf4 transformed host strain
  • Pichia pastoris GS115 (his4-) was purchased from Invitrogen, inoculated on YPD plates, and cultured at 30 ° C for 2 days to isolate monoclonals. The monoclonal was inoculated in 5 ml of YPD liquid medium, and cultured at 30 ° C until the OD was about 1.3-1.5. The cells were collected by centrifugation, rinsed with sterile cooling water, centrifuged, and suspended in 1 ml of ice-cold 1 M sorbitol solution.
  • Example 8 Screening of Mut + and Mut s transformants
  • the methylotrophic yeast contains two AOX genes, AOX1 and AOX2, respectively.
  • the expression vector is linearized with a different enzyme (e.g., BglII, Notl or Sail, Stul, etc.) and integrated into the position of the AOX1 or HIS4 gene of the yeast genome.
  • a Mut s phenotype methanol utilization slow
  • the wild-type yeast grows normally in methanol-containing medium and is called the Mut+ phenotype.
  • Example 7 Prepare MD-Sepharose plate and MM-Sepharose plate.
  • GS115- Albumin His ⁇ uf and GS 115-Gal (His+Mut + ) as controls
  • the transformants obtained in Example 7 were sequentially symmetrically inoculated from MM plate to MD plate with a sterile toothpick. The growth of the transformants was observed by culturing on both plates at 30 °C.
  • the Mut s phenotype grew slowly in methanol-containing medium and the Mut+ phenotype grew normally in methanol-containing medium, see Figure 9.
  • the transformant of His + Mut + was picked as a genetic engineering strain, named GS115-3EGF-E4orf4, and the expression of EGF-E4orf4 was observed.
  • Example 9 Expression of EGF-E4orf4
  • the genetically engineered strain GS115-3EGF-E4orf4 obtained in Example 8 was cultured in a shake flask. Inoculate the cells in a single or 80 °C culture in 25nil BMG medium, culture at 30 °C for 18-24 hours, and OD 6 (K) two 10 ⁇ 12. Collect the cells by centrifugation and transfer to 250ml BMMY medium. The culture was cultured at 30 ° C, and methanol was added at a concentration of 1% every 24 hours to induce expression. The optimal induction time was determined to be 96 to 120 h. The culture supernatant was collected by centrifugation, and the EGF-E4orf4 fusion protein was detected.
  • Example 10 Speculation of recombinant EGF-E4orf4 fusion protein
  • the recombinant EGF-E4orf4 fusion protein obtained in the above Example 9 was analyzed by SDS-PAGE protein electrophoresis. 15% separation using the Bio-Rad protein electrophoresis unit Gum, collected culture supernatant, added protein electrophoresis sample buffer, denatured at 100 ° C for 5 min, applied to electrophoresis, 100 V constant pressure, electrophoresis to bromophenol blue gel, stop electrophoresis, stain with silver nitrate.
  • the molecular weight of the EGF-E4orf4 fusion protein is about 20 kD, which is in accordance with the molecular weight of the EGF-E4orf4 fusion protein of amino acid composition.
  • Lane 1 GS115-3 EGF-E4orf4 expression supernatant, the arrow shows the EGF-E4orf4 fusion protein band.
  • Lane 2 Protein low molecular weight standards, the fragments from large to small are: 43kD, 29kD, 18.4kD, 14.3kD, 6.2kD.
  • EGF-E4orf4 fusion protein was first separated by SDS-PAGE and electrotransferred to a nitrocellulose membrane.
  • rabbit anti-human EGF polyclonal antibody purchased from Santa Cruz
  • rabbit anti-E4orf4 protein antiserum was added.
  • the inventors prepared according to the conventional method were incubated with a nitrocellulose membrane at room temperature for 1 hour, and then washed with HRP-labeled goat anti-rabbit IgG antibody at room temperature for 1 hour, rinsed and detected by ECL chemiluminescence.
  • EGF-E4orf4 fusion protein is positive for human EGF antibody.
  • Lane 11 Lane 1 : Protein low molecular weight standards, the fragments from large to small are: 43kD, 29kD, 18.4kD, 14.3kD, 6.2kD.
  • Lane 2-3 GS115-3 EGF-E4orf4 expression supernatant, primary antibody is rabbit anti-human EGF polyclonal antibody.
  • Lane 4 GS115_3EGF_E4orf4 expression supernatant, the primary antibody is rabbit anti-E4orf4 antiserum, and the arrow shows the EGF-E4orf4 fusion protein band.
  • Example 11 Isolation and purification of recombinant EGF-E4orf4 fusion protein
  • the yeast containing the recombinant EGF-E4orf4 fusion protein obtained in Example 9 was expressed and purified by cation exchange chromatography using a FPLC system of Pharmacia.
  • the yeast expression supernatant was diluted with 6-7 volumes of buffer A (50 mM NaAC/HAC, pH 4.0) and filtered through a 0.45 ⁇ microporous membrane; using 5 cylinders After accumulating the buffer A, the medium was loaded, the flow rate was 5 ml/min, and after the sample was washed to the baseline with buffer A, the flow rate was adjusted to 3 ml/min, and buffer B with different salt concentrations (bl: 0.5 M).
  • buffer A 50 mM NaAC/HAC, pH 4.0
  • the eluate containing the fusion protein identified by electrophoresis was collected, desalted by dialysis, concentrated by PEG-10000, sterilized by filtration through a 0.22 ⁇ m filter, stored at 20 ° C, and quantified by protein. See Figure 12.
  • Lane 1 Fusion protein EGF-E4orf4 yeast expression supernatant.
  • Lane 2 Protein low molecular weight standards, the fragments from large to small are: 43kD, 29kD, 18.4kD, 14.3kD.
  • Lane 3 Eluate collected by cation exchange chromatography, indicated by the arrow as recombinant EGF-E4orf4 fusion protein. Example 12.
  • the monolayer cultured cells were digested with 0.25% trypsin, and boiled into a single cell suspension with a medium containing 10% fetal bovine serum, and seeded in a 96-well culture plate at 5000 cells per well at a volume of 200 per well. ⁇ 1, 37 ° C, 5% C0 2 culture, 20-24 hours to be fine After the cells were completely adherent, the supernatant was discarded and replaced with medium containing 5% fetal bovine serum at 200 ⁇ , 37 ° C, 5% C0 2 per well.
  • the fusion protein EGF-E4orf4 obtained in Example 11 was serially diluted with PBS from the concentration of lng-15 g/ml, and the EGF standard was used as a control, and diluted according to the concentration of the fusion protein EGF-E4orf4, etc., per well.
  • BT325 cells human brain glioma cells, constructed by Beijing Tiantan Hospital
  • EGF-E4orf4 human brain glioma cells, constructed by Beijing Tiantan Hospital
  • the cell viability continued to decrease.
  • the fusion protein reached 12.5 ⁇ ⁇ / ⁇ 1
  • the highest concentration of the fusion protein used in this experiment the cell viability was minimized, and the OD 57Q was 0.183, which was far lower than the negative control group without sputum .
  • the activity was 0.276, which was completely different from the positive control cells in which EGF was added.
  • MDA-MB-231 cells human breast cancer cells, ATCC Number: HTB-26
  • EGF-E4orf4 fusion protein EGF-E4orf4
  • the cell viability was minimized from the concentration of 500 ng/ml.
  • Below the unstimulated control cell group at subsequent irritant concentrations (2.5 g/ml-15 g/ml), cell viability is always The control cell group was lower or closer to the unstimulated control group, and the cell viability of the EGF control group was always higher than that of the unstimulated group, indicating that the fusion protein EGF-E4orf4 can inhibit the activity of MDA-MB-231 cells.
  • Example 13 Flow cytometry for detection of apoptosis
  • apoptotic cells During the process of apoptosis, due to the concentration of cytoplasm and chromatin, the nucleus cleaves to produce apoptotic bodies, which leads to changes in the light scattering properties of the cells. Compared to living cells or necrotic cells, apoptotic cells have a special light scattering type - low forward scatter and high side scatter. Flow cytometry is capable of measuring changes in cell light scattering and is therefore widely used for the observation, detection and analysis of apoptosis.
  • the concentration of the fusion protein which is capable of substantially saturating the cell surface is used as the end point, and the concentration of the fusion protein which is capable of substantially saturating the cell surface is the starting point, in this range.
  • Set a series of continuous fusion protein concentrations (10 ⁇ 8 /3 ⁇ 10 5 cells - 25 ⁇ ⁇ /3 ⁇ 10 5 cells), co-culture with MDA-MB-231 cells, L15 medium containing 5% FBS, culture 72 Cellular changes were measured by flow cytometry after an hour. The results are shown in Figures 15-19. It can be seen that the fusion protein EGF-E4orf4 is capable of apoptotic MDA-MB-231 cells.
  • the degree of apoptosis is positively correlated with the concentration of the fusion protein.
  • apoptosis is also absent; the fusion protein rises from 10 ⁇ ⁇ to 25 ⁇ cells from a small amount of apoptosis to obvious apoptosis of the cells until the cells enter the late stage of apoptosis.
  • EGF-E4orf4 can induce apoptosis in MDA-MB-231 cells, and MDA-MB-231 cells are relatively sensitive to the cytotoxicity of the fusion protein. Apoptosis is evident.
  • MDA-MB-231 cells were cultured for 72 hours, and 92.1% of the cells grew well.
  • MDA-MB-231 cells were co-cultured with the fusion protein EGF-E4orf4 (10 g/3x10 5 cells), and a small number of cells had apoptosis, accounting for 15.4%.
  • MDA-MB-231 cells were co-cultured with the fusion protein (15 g/3x10 5 cells), and 28.2% of the cells were detached from the cell cycle, and the cells developed obvious apoptosis.
  • the characteristics of early cell apoptosis can be seen from the figure, that is, a slope generated by apoptotic cells or a sub-peak before the Go / G! peak. This is because in the early and middle stages of apoptosis, nuclear DNA is broken and is degraded into DNA fragments of integer multiples of nucleosomes. The arrow shows a subdiploid peak.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Public Health (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention décrit un facteur de croissance de l’épiderme humain de recombinaison - la protéine adénovirale de fusion E4orf4. L’invention décrit également un gène de fusion codant ladite protéine de fusion, un vecteur d’expression comprenant ce gène de fusion et un procédé utilisant de la levure méthylotrophe pour exprimer la protéine sécrétoire de fusion de recombinaison. En outre, la présente invention concerne également une combinaison de médicaments qui comprend ladite protéine et l'application thérapeutique de celle-ci.
PCT/CN2005/000211 2005-02-22 2005-02-22 Protéine de fusion antitumorale de ciblage comprenant la protéine adénovirale e4orf4 WO2006089455A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2005/000211 WO2006089455A1 (fr) 2005-02-22 2005-02-22 Protéine de fusion antitumorale de ciblage comprenant la protéine adénovirale e4orf4

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2005/000211 WO2006089455A1 (fr) 2005-02-22 2005-02-22 Protéine de fusion antitumorale de ciblage comprenant la protéine adénovirale e4orf4

Publications (1)

Publication Number Publication Date
WO2006089455A1 true WO2006089455A1 (fr) 2006-08-31

Family

ID=36927017

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2005/000211 WO2006089455A1 (fr) 2005-02-22 2005-02-22 Protéine de fusion antitumorale de ciblage comprenant la protéine adénovirale e4orf4

Country Status (1)

Country Link
WO (1) WO2006089455A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107904251A (zh) * 2017-12-28 2018-04-13 吉林大学 TAT‑hEGF融合蛋白的制备及其在隐形面膜的应用
CN113788898A (zh) * 2021-09-16 2021-12-14 陕西理工大学 BmSPI39的同型串联多聚体及其构建方法和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1293202A (zh) * 1999-10-13 2001-05-02 中国人民解放军农牧大学军事兽医研究所 包括修饰的导向部分的嵌合毒素

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1293202A (zh) * 1999-10-13 2001-05-02 中国人民解放军农牧大学军事兽医研究所 包括修饰的导向部分的嵌合毒素

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PENGLI ET AL.: "Expression of Epidermal Growth Factor-Adenovirus E4orf4 Fusion Protein in Tumor Cells and Its Cytotoxicity", CHINESE JOURNAL OF CANCER, vol. 24, no. 1, January 2005 (2005-01-01), pages 34 - 35 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107904251A (zh) * 2017-12-28 2018-04-13 吉林大学 TAT‑hEGF融合蛋白的制备及其在隐形面膜的应用
CN107904251B (zh) * 2017-12-28 2020-06-02 吉林大学 TAT-hEGF融合蛋白的制备及其在隐形面膜的应用
CN113788898A (zh) * 2021-09-16 2021-12-14 陕西理工大学 BmSPI39的同型串联多聚体及其构建方法和应用
CN113788898B (zh) * 2021-09-16 2022-09-23 陕西理工大学 BmSPI39的同型串联多聚体及其构建方法和应用

Similar Documents

Publication Publication Date Title
JP4411330B2 (ja) 腫瘍壊死因子関連リガンド
WO2023051701A1 (fr) Arnm, protéine et vaccin contre l'infection par sars-cov-2
JP2001514885A (ja) ７０個のヒト分泌タンパク質
JP2001514024A (ja) ５０個のヒト分泌タンパク質
JP2002505871A (ja) ３１個のヒト分泌タンパク質
JPH08501453A (ja) 外来遺伝子を送達し、発現させるベクター
WO2006089455A1 (fr) Protéine de fusion antitumorale de ciblage comprenant la protéine adénovirale e4orf4
US8030015B2 (en) Tumor-inhibiting protein and the use thereof
EP2128261B1 (fr) Adénovirus recombinant comprenant un gène khp53 recombinant et son procédé de préparation et ses utilisations
JP2003521215A (ja) ８３個のヒト分泌タンパク質
JPH04144684A (ja) エンドセリン受容体
JPH05103666A (ja) ヒト神経成長因子を発現させるための方法
WO2016158546A1 (fr) Composition d'antigène 6b du virus de l'herpès humain
CN113061168B (zh) 一种截短的发热伴血小板减少综合征病毒Gn蛋白及其应用
JP2790822B2 (ja) 組換リンホトキシン誘導体
CN111732667B (zh) 小反刍兽疫病毒基因工程亚单位疫苗
WO2011147138A1 (fr) Protéine de fusion de l'interleukine de ciblage et son procédé de préparation et son utilisation
WO2001060855A1 (fr) Nouvelle proteine humaine associee a la regulation du cycle cellulaire et sa sequence de codage
EP4029515A1 (fr) Effets anti-infectieux de la hnrnpa2b1, et utilisation de celle-ci
US20110288004A1 (en) Viral Sequences and Uses Thereof
JPH02485A (ja) 新規なヒトインターロイキン４、該因子を発現させるための組換えベクター及びそのベクターにより形質転換された形質転換体
CN100436481C (zh) 含腺病毒E4orf4蛋白的靶向性抗肿瘤融合蛋白
JP3803978B2 (ja) グルカゴン分解酵素及びそれをコードする遺伝子並びに該グルカゴン分解酵素に対する抗体
CN117229371A (zh) 新型冠状病毒变异毒株的S蛋白突变体及其基因工程化mRNA和疫苗组合物
JP2001512325A (ja) 改変型レチノブラストーマ腫瘍抑制タンパク質

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05714755

Country of ref document: EP

Kind code of ref document: A1

WWW Wipo information: withdrawn in national office

Ref document number: 5714755

Country of ref document: EP