WO2022053050A1 - 能破坏细胞的氨基酸序列及相关核苷酸序列和相关的应用 - Google Patents

能破坏细胞的氨基酸序列及相关核苷酸序列和相关的应用 Download PDF

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WO2022053050A1
WO2022053050A1 PCT/CN2021/118063 CN2021118063W WO2022053050A1 WO 2022053050 A1 WO2022053050 A1 WO 2022053050A1 CN 2021118063 W CN2021118063 W CN 2021118063W WO 2022053050 A1 WO2022053050 A1 WO 2022053050A1
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amino acid
acid sequence
mdpcd1
nucleotide sequence
seq
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French (fr)
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王月志
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王月志
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Priority to US18/245,095 priority Critical patent/US20230391836A1/en
Priority to CN202180006437.4A priority patent/CN114729021B/zh
Publication of WO2022053050A1 publication Critical patent/WO2022053050A1/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
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • 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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to an amino acid sequence capable of destroying cells, a nucleotide sequence encoding the corresponding amino acid, and related applications of the amino acid sequence and the nucleotide sequence.
  • ARF1 ADP-ribosylation factor 1
  • ARF1 ADP-ribosylation factor 1
  • ARF1 is conserved in its function and sequence characteristics in all eukaryotes (Cevher-Keskin, Int.J.Mol.Sci.2013, 14, 18181-18199).
  • ARF1 expression is associated with tumor cell replication and proliferation, and has been shown to be a molecular switch for cancer cell replication and proliferation (Boulay et al., The Journal of biological chemistry. 2008, 283:36425-36434; Hashimoto et al. ., Proceedings of the National Academy of Sciences of the United States of America. 2004, 101: 6647-6652; Schlienger et al., Oncotarget, 2016, 7: 11811-11827; Davis et al., Oncotarget, 2016, 7: 39834-39845).
  • ARF1 has been studied as a key molecular target for related cancer therapy and diagnosis (Schlienger et al., Oncotarget, 2016, 7:11811-11827; Davis et al., Oncotarget, 2016, 7:39834-39845; Ohashi et al.
  • Eukaryotic translation elongation factor eEF1 ⁇ is highly expressed and plays a key role in tumors (including breast cancer, ovarian cancer and lung cancer, etc.) and many human diseases (Abbas et al., Front. Oncol., 2015, 5:75).
  • Targeted inhibition of eEf1a by narcicycline can induce apoptosis of cancer cells and can effectively treat melanoma (Van Goietsenoven et al., FASEB J. 2010, 24(11):4575-84).
  • gene therapy has the potential to be highly efficient in both drug preparation and treatment. Therefore, at this time, it can interact with ARF1 or eEF1a to initiate the destruction of cells to achieve the effect of cancer treatment.
  • the present invention provides an amino acid sequence capable of destroying cells, a nucleotide sequence encoding the corresponding amino acid, and related applications of the amino acid sequence and the nucleotide sequence, so as to achieve the destruction of cells and provide a new solution for tumor therapy , To this end, the present invention provides the following technical solutions.
  • an amino acid sequence capable of destroying cells is provided, and the cell destroying means can trigger the collapse of the cell membrane system to achieve the effect of destroying cells, and may further include the effect of tissue damage caused by cell destruction.
  • amino acid sequence provided by the present invention also has such a feature: wherein, the amino acid sequence includes: the amino acid sequence shown in SEQ ID NO.: 1 and/or SEQ ID NO.: 29.
  • the amino acid sequence provided by the present invention also has such a feature: wherein, the amino acid sequence is the Mdpcd1-303 protein fragment with the amino acid sequence shown in SEQ ID NO.:2, or the amino acid residue of SEQ ID NO.:2 A derivative protein of the Mdpcd1-303 protein fragment derived from SEQ ID NO.:2 whose sequence has undergone substitution, deletion or addition of one or more amino acid residues and has the same activity as the amino acid residue sequence of SEQ ID NO.:2 or homologous proteins.
  • the amino acid sequence provided by the present invention also has the following characteristics: wherein the derived proteins include Mdpcd1-18 protein fragment, Mdpcd1-297 protein fragment and Mdpcd1-307 protein fragment, homologous proteins include Ptpcd1-296 protein fragment, Mdpcd1-18 protein fragment
  • the fragment has the amino acid sequence shown in SEQ ID NO.:3; the Mdpcd1-297 protein fragment has the amino acid sequence shown in SEQ ID NO.:4; the Mdpcd1-307 protein fragment has the amino acid sequence shown in SEQ ID NO.:30 , the Ptpcd1-296 protein fragment has the amino acid sequence shown in SEQ ID NO.:5.
  • a nucleotide sequence is provided for encoding an amino acid sequence capable of destroying cells, wherein the amino acid sequence is the above-mentioned amino acid sequence.
  • the nucleotide sequence provided by the present invention also has the feature that the nucleotide sequence is used to encode any one of the Mdpcd1-303 protein fragment, the derived protein of the protein fragment, and the homologous protein of the protein fragment.
  • the nucleotide sequence provided by the present invention also has the following characteristics: wherein, the nucleotide sequence is used to encode the Mdpcd1-303 protein fragment, and the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO.:6.
  • the nucleotide sequence provided by the present invention also has the following characteristics: wherein, the derived proteins of the Mdpcd1-303 protein fragment include the Mdpcd1-18 protein fragment and the Mdpcd1-297 protein fragment, and the homologous protein includes the Ptpcd1-296 protein fragment, wherein,
  • the nucleotide sequence used for coding the Mdpcd1-18 protein fragment is the nucleotide sequence shown in SEQ ID NO.:7
  • the nucleotide sequence used for coding the Mdpcd1-297 protein fragment is shown in SEQ ID NO.:8
  • the nucleotide sequence for encoding the Mdpcd1-307 protein fragment is the nucleotide sequence shown in SEQ ID NO.:31
  • the nucleotide sequence for encoding the Ptpcd1-296 protein fragment is SEQ ID The nucleotide sequence shown in NO.:9.
  • the present invention also provides a vector (preferably an expression vector) comprising: the above-mentioned nucleotide sequence.
  • the amino acid sequence, or the nucleotide sequence, or the application of the vector in destroying cells wherein the amino acid sequence is the above-mentioned amino acid sequence; the nucleotide is the above-mentioned nucleotide sequence ;
  • the carrier is the above-mentioned carrier.
  • the present invention also provides an amino acid sequence, or a nucleotide sequence, or the application of a vector in tumor treatment, characterized in that: wherein, the amino acid sequence is the above amino acid sequence; the nucleotide is the above nucleotide sequence; the vector is the above a.
  • the present invention also provides an amino acid sequence, or a nucleotide sequence, or the application of a vector in the preparation of a composition for destroying cells, wherein the amino acid sequence is the above-mentioned amino acid sequence; the nucleotide is the above-mentioned nucleoside acid sequence; the vector is the above-mentioned vector.
  • the present invention also provides an amino acid sequence, or a nucleotide sequence, or the application of a vector in the preparation of a drug for tumor treatment, wherein: the amino acid sequence is the above-mentioned amino acid sequence; the nucleotide is the above-mentioned nucleotide sequence sequence; the vector is the above-mentioned vector.
  • composition comprising: an amino acid sequence, or a nucleotide sequence, or a vector, wherein the amino acid sequence is the above-mentioned amino acid sequence; the nucleotide is the above-mentioned nucleotide sequence;
  • the carrier is the above-mentioned carrier.
  • the composition further contains a pharmaceutically acceptable carrier.
  • the present invention also provides a pharmaceutical composition, which is characterized by comprising: an amino acid sequence, or a nucleotide sequence, or a carrier, wherein the amino acid sequence is the above-mentioned amino acid sequence; the nucleotide is the above-mentioned nucleotide sequence; the carrier for the above-mentioned carrier.
  • a method for destroying cells comprising the steps of: (a) contacting the cells to be destroyed with the destructive polypeptide of claim 1, thereby causing the cell membrane system of the cells to collapse, thereby destroying the cells.
  • the cells are mammalian cells.
  • the cells are tumor cells.
  • step (a) a nucleic acid or a vector expressing the destructive polypeptide is introduced into the cell, so that the destructive polypeptide is expressed or overexpressed in the cell.
  • the method further comprises the step of: (b) detecting the integrity of the cell membrane of the cells in step (a) and/or whether the cells survive, so as to qualitatively or quantitatively determine the destruction of the cells .
  • the method is non-therapeutic and non-diagnostic.
  • the method is an in vitro method.
  • the method is therapeutic.
  • the present invention obtains Mdpcd1-303 with the amino acid sequence shown in SEQ ID NO.: 1 and/or SEQ ID NO.: 29 and its derived proteins or homologous proteins, which have been confirmed to trigger the breakdown of the cell membrane system , to achieve the effect of destroying cells, and it has been confirmed that the nucleotides encoding the amino acids of this sequence can have a therapeutic effect on tumors, and the corresponding amino acids can also have a therapeutic effect on tumors.
  • the protein fragment Mdpcd1-303 or its derivative protein fragment Mdpcd1-18 or its derivative protein fragment Mdpcd1-297 provided by the present invention is prepared by using any expression vector that can guide the appropriate expression of foreign genes in tumor cells, including overexpression.
  • the encoding nucleotide sequence of its derivative protein fragment Mdpcd1-307 or its homologous protein fragment Ptpcd1-296 can be introduced into tumor cells, which can change the life process of tumor cells, initiate tumor cell programmed death, and realize the inhibition and killing of tumor tissue , to enhance the immune function of the body.
  • any enhanced promoter, inducible promoter or tumor cell-specific promoter can be added before the transcription initiation nucleotide.
  • the expression vector containing the above-mentioned nucleotide sequence of the present invention can be used as a gene transfer system to transfect tumor cells by using various viral vectors such as adenovirus, retrovirus, adeno-associated virus, vaccinia virus, herpes virus, lentivirus, etc. Use naked plasmid DNA, liposomes, cationic polymers and other methods to transfect tumor cells.
  • FIG. 1 is a picture of the results of the dip test of the transient expression vector of the nucleotide sequence of the Mdpcd1-303 protein fragment involved in Example 9.
  • FIG. 1 is a picture of the results of the dip test of the transient expression vector of the nucleotide sequence of the Mdpcd1-303 protein fragment involved in Example 9.
  • FIG. 2 is a picture of the results of the dip-infection test of the transient expression vector of the nucleotide sequence of the Mdpcd1-18 protein fragment involved in Example 9.
  • FIG. 2 is a picture of the results of the dip-infection test of the transient expression vector of the nucleotide sequence of the Mdpcd1-18 protein fragment involved in Example 9.
  • FIG. 3 is a picture of the results of the dip-infection test of the transient expression vector of the nucleotide sequence of the Mdpcd1-297 protein fragment involved in Example 9.
  • FIG. 3 is a picture of the results of the dip-infection test of the transient expression vector of the nucleotide sequence of the Mdpcd1-297 protein fragment involved in Example 9.
  • FIG. 4 is a picture of the results of the dip-infection test of the transient expression vector of the nucleotide sequence of the Ptpcd1-296 protein fragment involved in Example 9.
  • FIG. 4 is a picture of the results of the dip-infection test of the transient expression vector of the nucleotide sequence of the Ptpcd1-296 protein fragment involved in Example 9.
  • FIG. 5 shows the changes of tobacco leaf cells after the transient expression vector of the nucleotide sequence of the Mdpcd1-303 protein fragment involved in Example 9 was infiltrated.
  • FIG. 6 is a picture of the results of the dip-infection test of the transient expression vector of the Atpcd1 protein nucleotide sequence involved in Example 9.
  • FIG. 6 is a picture of the results of the dip-infection test of the transient expression vector of the Atpcd1 protein nucleotide sequence involved in Example 9.
  • FIG. 7 is the verification of the yeast two-hybrid protein interaction between Mdpcd1-303 protein involved in Example 10 and ARF1 (QDO medium).
  • Figure 8 is the subcutaneous tumor growth curve of Example 11 Adc68-Mdpcd1-303 injection treatment and control tumor-bearing mice. **P ⁇ 0.01.
  • Figure 9 is the subcutaneous tumor growth curve of Example 12 Adc68-Mdpcd1-18 injection treatment and control tumor-bearing mice. **P ⁇ 0.01.
  • Figure 10 is the subcutaneous tumor growth curve of Example 13 Adc68-Mdpcd1-297 injection treatment and control tumor-bearing mice. **P ⁇ 0.01.
  • Figure 11 is the subcutaneous tumor growth curve of Example 14 Adc68-Ptpcd1-296 injection treatment and control tumor-bearing mice. **P ⁇ 0.01.
  • Example 12 is a picture of the results of the dip-infection test of the transient expression vector for the nucleotide sequence of the Mdpcd1-307 protein fragment involved in Example 17.
  • FIG. 13 is a flow cytometry cell apoptosis detection result of the Mdpcd1-307 protein involved in Example 18 on the viability inhibition effect of SMMC-7721 (human hepatoma cells, the same below).
  • FIG. 14 shows the results of the detection of the inhibition of the infectivity of SMMC-7721 by the Mdpcd1-307 protein involved in Example 18.
  • FIG. 15 is the TUNEL staining detection result of the Mdpcd1-307 protein involved in Example 19 promoting the apoptosis rate of SMMC-7721 tumor cells.
  • Figure 16 shows the number of differentially expressed genes in SMMC-7721 tumor regulated by Mdpcd1-307 protein involved in Example 19.
  • FIG. 17 is a functional enrichment cluster of metabolic pathways of differentially expressed genes in SMMC-7721 tumor regulated by Mdpcd1-307 protein involved in Example 19.
  • FIG. 17 is a functional enrichment cluster of metabolic pathways of differentially expressed genes in SMMC-7721 tumor regulated by Mdpcd1-307 protein involved in Example 19.
  • Figure 18 shows the tumor inhibition of mouse SMMC-7721 by Mdpcd1-307 protein involved in Example 19.
  • the inventors After extensive and in-depth research, the inventors have unexpectedly developed a destructive polypeptide derived from plants with unique functions for the first time. When the destructive polypeptide is overexpressed, it can trigger the collapse of the cell membrane system and achieve the effect of destroying cells.
  • the destructive polypeptide Mdpcd1-303 of the present invention is derived from plants, and is a plant-specific gene, and there is no homologous gene in animals; the protein encoded by this gene can cause tobacco when it is overexpressed (for example, through the 35S promoter to promote gene expression). Leaf cells die.
  • the study of the present invention also shows that the interacting proteins (such as ARF1 or eEF1a) of Mdpcd1-303-encoded proteins play a key role in the tobacco leaf cell death initiated by this gene; ARF1 and eEF1a are conserved in animals and plants, and It is closely related to the occurrence of various tumors.
  • ARF1 and eEF1a are conserved in animals and plants, and It is closely related to the occurrence of various tumors.
  • Mdpcd1-307 is an allelic variant of Mdpcd1-303.
  • the protein encoded by Mdpcd1-307 cannot cause tobacco leaf cell death when overexpressed (expressed by the 35S promoter), but when Mdpcd1-307 is co-expressed with ARF1, can cause tobacco leaf cell death; similarly, when Mdpcd1-307 is co-expressed with eEF1a, it can also cause tobacco leaf cell death.
  • Mdpcd1-303 can significantly increase the apoptosis rate of liver cancer cells; mouse in vivo experiments show that Mdpcd1-303 can significantly inhibit the growth of tumor tissue.
  • the allele or core sequence of Mdpcd1-303 also has a similar function.
  • administration refers to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, peritoneal Intra, spinal or other parenteral routes of administration, such as by injection or infusion.
  • disrupting polypeptide As used herein, the terms “disrupting polypeptide”, “polypeptide of the invention”, “disrupting polypeptide of the invention” are used interchangeably to refer to Mdpcd1-303 and its derived or homologous proteins. It is to be understood that the term includes wild type or mutant type. In addition, the term also includes full-length proteins or functional fragments thereof.
  • a preferred disruptive protein is Mdpcd1-303 and its derivative proteins (such as Mdpcd1-18, Mdpcd1-297, Mdpcd1-307 having the amino acid sequence shown in SEQ ID NO.: 1 and/or SEQ ID NO.: 29) ) or homologous proteins (eg Ptpcd-296).
  • the experiments of the present invention show that the overexpressed protein or the presence of a large amount can trigger the collapse of the cell membrane system to achieve the effect of destroying cells, and it has been confirmed that the nucleotides encoding the amino acids in this sequence can have a therapeutic effect on tumors, and the corresponding amino acids also Can have a therapeutic effect on tumors.
  • the protein fragment Mdpcd1-303 or its derivative protein fragment Mdpcd1-18 or its derivative protein fragment Mdpcd1- 297 or its derivative protein fragment Mdpcd1-307 or its homologous protein fragment Ptpcd1-296 encoding nucleotide sequence into tumor cells can change the life process of tumor cells, start tumor cell programmed death, and realize the inhibition and destruction of tumor tissue. Kill, enhance the immune function of the body.
  • destroying a cell or “destroying a cell” are used interchangeably and refer to the effect of triggering the collapse of the cell membrane system to the effect of destroying the cell, and may further include the effect of tissue damage caused by the destruction of the cell.
  • the destroying cells refers to causing destruction or damage to plant cells, animal cells, cancer cells, etc., or a combination thereof, or their corresponding tissue structures.
  • the cells include (but are not limited to): plant cells and animal cells.
  • plant cells and animal cells Preferably, human and non-human mammalian cells.
  • the cells are diseased cells, such as tumor cells.
  • the tumor cells include (but are not limited to): ovarian cancer cells, lung cancer cells, pancreatic cancer cells, liver cancer cells, gastric cancer cells, breast cancer cells, nasopharyngeal cancer cells, esophageal cancer cells, Colorectal cancer cells, cervical cancer cells, leukemia and lymphoma cells, etc.
  • the destroying cells refers to destroying and injuring tobacco leaf cells and tobacco leaves.
  • the destroying cells refers to causing destruction and damage to liver cancer cells and hepatocellular carcinoma cells.
  • the present invention also provides a vector comprising the amino acid sequence or nucleotide sequence of the present invention.
  • Vectors derived from retroviruses such as adenoviruses, are suitable tools for long-term gene transfer because they allow long-term, stable integration of the transgene into the cell genome and replication with the replication of daughter cell genomes.
  • Adenoviral vectors have high transgenic efficiency, and in vitro experiments are usually close to 100% transduction efficiency; different types of human tissue cells can be transduced, regardless of whether the target cells are dividing cells or not; high titer viral vectors can be easily prepared; enter cells It is not integrated into the host cell genome, but is only expressed instantaneously, with high safety. Therefore, adenoviral vectors have more and more applications in gene therapy clinical trials, and become the most widely used and most promising viral vectors after retroviral vectors.
  • amino acid sequence or nucleotide sequence of the present invention can be ligated downstream of a promoter and incorporated into an expression vector by routine procedures.
  • the vector can integrate into the eukaryotic genome and replicate therewith.
  • Typical cloning vectors contain transcriptional and translational terminators, initial sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the expression vectors of the present invention can also be used in standard gene delivery protocols for nucleic acid immunization and gene therapy. Methods of gene delivery are known in the art. See, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety.
  • the amino acid sequence or nucleotide sequence can be cloned into many types of vectors.
  • the amino acid sequence or nucleotide sequence can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Particular vectors of interest include expression vectors, replication vectors, and the like.
  • expression vectors can be provided to cells in the form of viral vectors.
  • Viral vector techniques are well known in the art and are described, for example, in Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory, New York, 2001) and other virology and molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • suitable vectors contain at least one origin of replication functional in the organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many DNA virus systems are known in the art.
  • Many adenoviral vectors are known in the art.
  • promoter elements can regulate the frequency of transcription initiation.
  • these elements are located in the 30-110 bp region upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site.
  • the spacing between promoter elements is often flexible so that promoter function is maintained when an element is inverted or moved relative to another element.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased by 50 bp before activity begins to decline.
  • individual elements appear to act cooperatively or independently to initiate transcription.
  • a suitable promoter is the cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is elongation growth factor-1a (EF-1a).
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse breast cancer virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus (EBV) immediate early promoter, Russell sarcoma virus promoter, and human gene promoters such as but not limited to actin protein promoter, myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.
  • an inducible promoter provides a molecular switch that enables expression of the polynucleotide sequence linked to the inducible promoter to be turned on when desired, or turned off when not required.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • Expression vectors introduced into cells may also contain either or both selectable marker genes or reporter genes to facilitate identification and selection of expressing cells from transfected or infected cell populations by viral vectors.
  • the selectable marker can be carried on a single piece of DNA and used in co-transfection procedures. Both the selectable marker gene and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable marker genes include, for example, antibiotic resistance genes such as neomycin and the like.
  • an expression vector can be readily introduced into a host cell, eg, mammalian (eg, human T cells), bacterial, yeast, or insect cells, by any method known in the art.
  • a host cell eg, mammalian (eg, human T cells), bacterial, yeast, or insect cells
  • an expression vector can be transferred into a host cell by physical, chemical or biological means.
  • Physical methods of introducing polynucleotides into host cells include calcium phosphate precipitation, cationic complex transfection, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, eg, Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor Laboratory, New York, 2001). Preferred methods for introducing polynucleotides into host cells are liposome transfection and cationic complex polyethyleneimine transfection.
  • Biological methods for introducing polynucleotides into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, eg, human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, among others. See, eg, US Patent Nos. 5,350,674 and 5,585,362.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
  • Exemplary colloidal systems for use as in vitro and in vivo delivery vehicles are liposomes (eg, artificial membrane vesicles).
  • exemplary delivery vehicles are liposomes.
  • lipid formulations is contemplated to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • nucleic acid can be associated with a lipid.
  • Nucleic acids associated with lipids can be encapsulated into the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached via linker molecules associated with both liposomes and oligonucleotides to liposomes, entrapped in liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complex with micelles, or otherwise associated with lipids.
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. They can also simply be dispersed in solution, possibly forming aggregates that are not uniform in size or shape.
  • Lipids are lipid substances, which can be naturally occurring or synthetic lipids.
  • lipids include lipid droplets, which occur naturally in the cytoplasm and in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.
  • the vector is an adenovirus vector.
  • the present invention also provides a pharmaceutical composition containing any molecular entity that promotes the expression or enhanced activity of the cell-destroying amino acid sequence and/or nucleotide sequence, or contains the cell-destroying amino acid sequence and/or Or nucleotide sequences, or expression vectors that promote molecular entities capable of disrupting the expression and enhanced activity of cell-destructing amino acid sequences and/or nucleotide sequences, and other pharmaceutically acceptable vectors.
  • the pharmaceutical composition of the present invention usually contains adenovirus particles of 10 8 -10 9 PFU.
  • the term "pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. They are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, buffers. In addition, auxiliary substances such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The carrier may also contain cell transfection reagents.
  • the pharmaceutical composition of the present invention can be obtained after mixing the expression vector and a pharmaceutically acceptable carrier.
  • the mode of administration of the composition of the present invention is not particularly limited, and representative examples include but are not limited to: intravenous injection, subcutaneous injection, brain injection, intrathecal injection, and spinal cord injection.
  • the molecular entity described in the present invention contains the amino acid sequence and/or nucleotide sequence capable of destroying cells with enhanced expression or activity, or contains the amino acid sequence and/or nucleotide sequence capable of destroying cells, or promotes energy
  • the expression vector with enhanced expression and activity of the amino acid sequence and/or nucleotide sequence of the destroyed cell can be used to prepare the destroyed tumor cell, inhibit its replication and proliferation, change the life process of the tumor cell, initiate the programmed cell death of the tumor cell, and realize the anti-tumor effect on the tumor tissue. Drugs that inhibit and kill, and enhance the body's immunity.
  • the protein encoded by any amino acid sequence and/or nucleotide sequence that can promote the cell destruction described in the present invention has an interaction relationship with the GTPase family (such as ARF1 or eEF1a), and the enzyme
  • the gene variation in the class family is related to the occurrence of various cancers, so the present invention can also be used to prepare a potential drug for the treatment of cancer related to the GTPase class gene variation.
  • the present invention has obtained the amino acid sequence shown in SEQ ID NO.: 1 and/or SEQ ID NO.: 29, the nucleotide sequence encoding the amino acid sequence, and the amino acid sequence with SEQ ID NO.: 1 and/or SEQ ID NO.: 1 and/or SEQ ID NO.: 1.
  • the Mdpcd1-303 gene is derived from plants and is a plant-specific gene, and there is no homologous gene in animals.
  • the protein encoded by this gene can cause cell death when it is overexpressed (the gene expression is promoted by the 35S promoter), and the interacting proteins (such as ARF1 or eEF1a) of the Mdpcd1-303 encoded protein play a key role in the cell death initiated by this gene.
  • ARF1 and eEF1a are conserved in animals and plants, and are closely related to the occurrence of various tumors, the amino acid sequence shown in SEQ ID NO.: 1 and/or SEQ ID NO.: 29 described in the present invention.
  • the nucleotide sequence encoding the amino acid sequence and the vector including the nucleotide sequence can induce cell death by interacting with ARF1 and eEF1a in animals and plants, and expand the scope of action of plant-specific genes to animals.
  • Mdpcd1-307 is an allelic variant of Mdpcd1-303.
  • the protein encoded by Mdpcd1-307 cannot cause cell death when overexpressed (expressed by the 35S promoter), but when Mdpcd1-307 is co-expressed with ARF1, can cause cell death; similarly, when Mdpcd1-307 is co-expressed with eEF1a, it can also cause cell death. It further indicates that the interaction with ARF1 and eEF1a is a key factor in triggering cell death, and the encoded proteins that cannot cause cell death can be co-expressed to achieve the effect of triggering cell death.
  • the present invention obtains Mdpcd1-303 with the amino acid sequence shown in SEQ ID NO.: 1 and/or SEQ ID NO.: 29 and its derivative proteins or homologous proteins, which have been confirmed to trigger the collapse of the cell membrane system, achieve the effect of destroying cells. It can significantly increase the apoptosis rate of tumor cells and significantly inhibit the growth of tumor tissue, thereby having a therapeutic effect on tumors.
  • amino acid sequence shown in SEQ ID NO.: 1 and/or SEQ ID NO.: 29 obtained by the present invention and the nucleotides encoding the amino acids of the sequence can also have a therapeutic effect on tumors.
  • the allele or core sequence of Mdpcd1-303 also has a similar function.
  • the protein fragment Mdpcd1-303 provided by the present invention or its derivative protein fragment Mdpcd1-18 or its derivative protein fragment Mdpcd1- 297 or its derived protein fragment Mdpcd1-307 or its homologous protein fragment Ptpcd1-296 encoding nucleotide sequence into tumor cells can change the life process of tumor cells, start tumor cell programmed death, achieve inhibition of tumor tissue and Kill, enhance the immune function of the body.
  • GTPase GTPase family
  • ARF1 or eEF1a GTPase family
  • Gene variation in the enzyme family is related to the occurrence of various cancers, so the present invention can also be used to prepare a potential drug for cancer treatment related to GTPase gene variation.
  • SEQ ID NO.: 10 P1 upstream primer nucleotide sequence
  • SEQ ID NO.: 11 P1 downstream primer nucleotide sequence
  • SEQ ID NO.: 12 P2 upstream primer nucleotide sequence
  • SEQ ID NO.: 13 P2 downstream primer nucleotide sequence
  • SEQ ID NO.: 14 P3 upstream primer nucleotide sequence
  • SEQ ID NO.: 15 P3 downstream primer nucleotide sequence
  • SEQ ID NO.: 16 P4 upstream primer nucleotide sequence
  • SEQ ID NO.: 17 (P4 downstream primer nucleotide sequence)
  • SEQ ID NO.: 18 P5 upstream primer nucleotide sequence
  • SEQ ID NO.: 19 P5 downstream primer nucleotide sequence
  • SEQ ID NO.: 20 (P6 upstream primer nucleotide sequence)
  • SEQ ID NO.: 21 (P6 downstream primer nucleotide sequence)
  • SEQ ID NO.: 22 P7 upstream primer nucleotide sequence
  • SEQ ID NO.: 23 P7 downstream primer nucleotide sequence
  • SEQ ID NO.: 24 P8 upstream primer nucleotide sequence
  • SEQ ID NO.: 25 (P8 downstream primer nucleotide sequence)
  • SEQ ID NO.: 26 P9 upstream primer nucleotide sequence
  • SEQ ID NO.: 27 (P9 downstream primer nucleotide sequence)
  • the coding nucleotide sequence of the target protein fragment was obtained by PCR amplification
  • RNA of the corresponding experimental materials was extracted with the Plant Trizol kit from Yingjun Company, and the quality of the total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.
  • Promega's reverse transcription kit was used for reverse transcription, single-stranded cDNA was synthesized as a template, and the designed primers were used to amplify the target fragment.
  • the PCR reaction system was 25 ⁇ L, containing 5 ng of template, 5 pmol of F and R primers, 2.5 ⁇ L of 10 ⁇ PCR buffer, 37.3 nmol of MgCl 2 , 5 nmol of dNTP, and 0.5 U of rTaq polymerase.
  • the amplification program was as follows: pre-denaturation at 94°C for 3 min; 30 cycles of 94°C for 20s, 60°C for 30s, and extension at 72°C for 60s; and 72°C for 5 min.
  • the primers were designed to introduce restriction sites, and the obtained target coding nucleotide sequences were used as templates for PCR amplification, and the PCR products were digested, purified and quantified, and cloned into the EcoRI and BamHI restriction sites of the expression vector PET-32a.
  • Click, convert E.coli. Pick a single colony and shake the bacteria overnight in 1 mL of LB (Amp 100 g/mL), transfer to 200 mL of fresh LB medium and shake until the bacterial concentration A600 ⁇ 0.6; add IPTG to a final concentration of 1.0 mM, and culture at 37 °C for induction Express for 3 hours.
  • the bacterial solution was centrifuged at 12,000g for 5min, and the precipitate was suspended in extraction buffer (3M NaCl, 1mM PMSF, 50mM pH8.0 phosphate buffer) to sonicate the cells, centrifuged at 12,000g for 20min, and the supernatant was collected.
  • Equilibrate Ni-Sepharose gel with 10 mM imidazole, 50 mM pH8.0 phosphate buffer; add cell lysate for 20 min at room temperature, wash 3 times with 5 times the gel volume of equilibration buffer; then use 300 mM imidazole, 50 mM pH8.0 Phosphate buffer was used for elution, and the collected eluate was the purified Trx-expressed protein.
  • primers for P1 are as follows:
  • RNA obtained from young apple (Malus) leaves was extracted with the Plant Trizol kit of handsome company, followed by the subsequent steps of obtaining the nucleotide sequence as described in the general method, and amplified by PCR to obtain a nucleotide of 912 bp sequence, the PCR product was cloned into pMD18-T vector, and the nucleotide sequence obtained by sequencing was shown in SEQ ID NO.: 6.
  • the nucleotide sequence encodes 303 amino acids, and the amino acid sequence is shown in SEQ ID NO.:2.
  • the nucleotide sequence SEQ ID NO.: 7 of the peptide segment Mdpcd1-18 was synthesized by gene synthesis technology.
  • the sequence encodes 18 amino acids, and the amino acid sequence is shown in SEQ ID NO.:3.
  • the P2 primers are as follows:
  • RNA obtained from young apple (Malus) leaves was extracted with the Plant Trizol kit of handsome company, followed by the subsequent steps of obtaining the nucleotide sequence as described in the general method, and amplified by PCR to obtain a nucleotide of 912 bp sequence, the PCR product was cloned into the pMD18-T vector, and the nucleotide sequence obtained by sequencing was shown in SEQ ID NO.: 8.
  • the nucleotide sequence encodes 297 amino acids, and the amino acid sequence is shown in SEQ ID NO.:4.
  • P3 primers Referring to the published genome sequence of P. trichocarpa, use Macvector software to design primer P3.
  • the P3 primers are as follows:
  • RNA was extracted from the young leaves of Populus trichocarpa with the Plant Trizol kit of handsome company, and the subsequent steps of obtaining the nucleotide sequence as described in the general method were used to amplify by PCR to obtain 891bp.
  • Nucleotide sequence, the PCR product was cloned into pMD18-T vector, and the sequence obtained by sequencing was shown in SEQ ID NO.: 9. This sequence encodes 296 amino acids, and the amino acid sequence is shown in SEQ ID NO.:5.
  • the obtained Mdpcd1-303 coding sequence SEQ ID NO.: 6 is a template to carry out PCR amplification, and the following steps of obtaining the target protein fragment described in the general method are adopted to obtain the purified apple Mdpcd1 protein fragment Mdpcd1-303.
  • the Mdpcd1-18 coding sequence SEQ ID NO.: 7 with both ends connected to the EcoRI and BamHI restriction sites was obtained by gene synthesis, and the gene synthesis product was subjected to the subsequent steps of obtaining the target protein fragment as described in the general method to obtain purification.
  • the protein fragment Mdpcd1-18 is a small molecule peptide, it can also be directly synthesized by peptide synthesis technology.
  • the primer P5 was designed to introduce the restriction site.
  • the P5 primers are as follows:
  • the obtained Mdpcd1 nucleotide sequence SEQ ID NO.: 8 is a template to carry out PCR amplification, and the subsequent steps of obtaining the target protein fragment described in the general method are used to obtain a purified protein fragment Mdpcd1-297.
  • the primer P6 was designed to introduce the restriction site.
  • the primers for P6 are as follows:
  • the obtained Mdpcd nucleotide sequence SEQ ID NO.: 9 is a template to carry out PCR amplification, and the subsequent steps of obtaining the target protein fragment described in the general method are used to obtain a purified protein fragment Ptpcd1-296.
  • SEQ ID NO.: 1 and/or SEQ ID NO.: 29 are conserved functional regions.
  • the coding sequences of the proteins of the above examples were constructed into transient expression vectors, and the tobacco leaves were respectively subjected to a 72-hour dip test, while the vector was empty as a control.
  • Atpcd1 protein coding sequence transient expression vector homologous to the Mdpcd1-303 protein was also used to carry out the same dip test.
  • the Atpcd1 protein amino acid sequence is shown in SEQ ID NO.: 28, and the sequence is not obtained in the above-mentioned respective embodiments.
  • the conserved sequence of the protein SEQ ID NO.: 1 and/or SEQ ID NO.: 29.
  • Fig. 1 is the result picture of the dip test of the Mdpcd1-303 protein fragment coding sequence transient expression vector involved in embodiment 9;
  • Fig. 2 is the result picture of the dip test of the Mdpcd1-18 protein fragment coding sequence transient expression vector involved in embodiment 9;
  • Fig. 3 is the result picture of the dip test of the Mdpcd1-297 protein fragment coding sequence transient expression vector involved in Example 9;
  • FIG. 4 is a picture of the results of the dip-infection test of the transient expression vector for the coding sequence of the Ptpcd1-296 protein fragment involved in Example 9.
  • FIG. 4 is a picture of the results of the dip-infection test of the transient expression vector for the coding sequence of the Ptpcd1-296 protein fragment involved in Example 9.
  • Figure 5 shows the changes of tobacco leaf cells after the transient expression vector of the coding sequence of the Mdpcd1-303 protein fragment involved in Example 9 was immersed in the experiment.
  • the damaged tobacco leaves (area A) after the transient expression vector of the Mdpcd1-303 protein fragment coding sequence were placed in the cell field of view, and the Mdpcd1-303-GFP fusion protein located on the cell membrane was accompanied by cell membrane rupture.
  • GF GFP fluorescence position is the cell membrane position, GFP fluorescence signal is the localization information of Mdpcd1-303; BF: cell membrane disintegration image under white light; Merged: the overlap of BF and GF after overlapping , which can indicate that Mdpcd1-303-GFP is localized to the cell membrane, and diffusely decomposes with the rupture of the cell membrane), indicating that the Mdpcd1-303 protein fragment is destructive to cells. From Figures 2-5, it is also speculated that derived or homologous fragments thereof are also destructive to cells.
  • FIG. 6 is a picture of the results of the dip-infection test of the transient expression vector of the Atpcd1 protein coding sequence involved in Example 9.
  • FIG. 6 is a picture of the results of the dip-infection test of the transient expression vector of the Atpcd1 protein coding sequence involved in Example 9.
  • FIG. 7 is the verification of the yeast two-hybrid protein interaction between Mdpcd1-303 protein involved in Example 10 and ARF1 (QDO medium).
  • Mdpcd1 bait plasmid and AFR1 prey plasmid recombinant plasmid were constructed.
  • the constructed bait plasmid and prey plasmid were extracted in large quantities and detected by agarose gel electrophoresis.
  • the ARF1 protein has sequence and functional conservation across species.
  • the apple ARF1 sequence (LOC103404322) used in the experiment was obtained from a public database and shared consensus.
  • the obtained bait plasmid was tested for toxicity and self-activation, and the bait plasmid and the prey were co-transformed into NMY51 competent cells.
  • the ability to grow on the DDO plate indicates that the recombinant bait plasmid was successfully transferred into the host bacteria and was non-toxic to the host bacteria.
  • TDO and QDO could not grow, indicating that the bait protein could not activate reporter gene expression.
  • the bait plasmid and plasmid POST-NubaI were co-transformed into NMY51 competent cells.
  • the coated plate DDO could grow, and the co-transformation was successful.
  • Both the coated TDO and QDO had colony growth, indicating that the reporter genes HIS and ADE2 were activated, indicating that the reading frame constructed by bait
  • the box is correct, the ubiquitin experimental system can function.
  • Yeast cells were co-transformed with the prey plasmid and the bait plasmid.
  • the control and functional verification results were in line with expectations, indicating that the system could be used for two-hybrid verification. can grow, indicating that there is an interaction between the two ( Figure 7).
  • This example illustrates that the protein fragment having the amino acid sequence SEQ ID NO.: 1 and/or SEQ ID NO.: 29 may cause damage to cells through the existing interaction with ARF1.
  • Mdpcd303 action in animals and plants Mdpcd1-303, etc. have amino acid sequences of SEQ ID NO.: 1 and/or SEQ ID NO.: 29
  • SEQ ID NO.: 1 and/or SEQ ID NO.: 29 The protein fragments mediated by cell programming Death is conserved in animals and plants, and the proliferation inhibition, killing and death of diseased cells such as tumors mediated by these proteins are widespread.
  • This embodiment is one of many similar functional application implementations of Mdpcd1-303 protein fragments with amino acid sequence SEQ ID NO.: 1 and/or SEQ ID NO.: 29.
  • Mdpcd1-303 recombinant adenovirus construction and cell packaging were completed by commercialization technical services provided by biotechnology companies.
  • the expected amplified fragment is 940bp, and the PCR reaction conditions are: 94°C for 3 min; 94°C for 20s, 58°C for 30s, 72°C for 1min, 30 cycles; 72°C for 7min. After separation by 1% agarose gel electrophoresis, the gel was cut and recovered.
  • the transfer plasmid pShuttle-CMV and the target gene Mdpcd1 were digested and purified by KpnI and XbaI, and the two were ligated by T4 DNA ligase.
  • Mdpcd1-303 was directionally cloned into the transfer plasmid pShuttle-CMV, and then digested and sequenced to verify the correctness of the clone.
  • coli stab-2 by the calcium chloride method, plated on LB plate (ampicillin) for screening, cloned, shaken, plasmid extraction, and the plasmid was purified by Bagl II , Xhol I, Mun I digestion and sequencing were correct.
  • the recombinant positive clone pShuttle-CMV-Mdpcd1-303-pAdc68 was successfully constructed.
  • HEK293 cells When HEK293 cells grew to about 80% confluence, pShuttle-CMV-Mdpcd1-303-pAdc68 was linearized by PacI digestion, and HEK293 cells were transfected by X-treme method, and the virus was packaged in HEK293 cells, cultured for 12 days, under light microscope See that the cells become round, fall off the bottom wall of the culture flask, and the nucleus occupies most of the cell volume, that is, cytopathic effect (CPE) occurs, collect the cells, centrifuge at 3,500 r/min for 5 min, remove the supernatant and add anti-serum-free DMEM to reconstitute the cells.
  • CPE cytopathic effect
  • Adc68- Mdpcd1-303 an adenovirus vector with the Mdpcd1-303 gene (named Adc68- Mdpcd1-303), stored at -80°C for later use.
  • Figure 8 is the subcutaneous tumor growth curve of Example 11 Adc68-Mdpcd1-303 injection treatment and control tumor-bearing mice. **P ⁇ 0.01.
  • Adc68-Mdpcd1-303 recombinant virus has obvious protective effect on the growth of nude mouse xenograft model.
  • Mdpcd1-18 Recombinant Adenovirus and Cell Packaging According to Example 11, the coding nucleotide sequence of Mdpcd1-18 was artificially synthesized, and KpnI and XbaI restriction sites were added to both ends of the coding nucleotide sequence during synthesis.
  • Figure 9 is the subcutaneous tumor growth curve of Example 12 Adc68-Mdpcd1-18 injection treatment and control tumor-bearing mice. **P ⁇ 0.01.
  • Adc68-Mdpcd1-18 could significantly inhibit tumor growth (Figure 9).
  • survival time the death of the nude mice in the Adc68-Mdpcd1-18 treatment group occurred after 40 days of treatment, however, all the mice in the control group died within 40 days.
  • the average lifespan of nude mice in the Adc68-Mdpcd1-18 group was significantly prolonged, with about half of the mice surviving for more than 60 days.
  • Adc68-Mdpcd1-18 recombinant virus has obvious protective effect on the growth of nude mice xenograft model.
  • Example 11 for the construction and cell packaging of Mdpcd1-297 recombinant adenovirus.
  • Figure 10 is the subcutaneous tumor growth curve of Example 13 Adc68-Mdpcd1-297 injection treatment and control tumor-bearing mice. **P ⁇ 0.01.
  • the mouse hepatocellular carcinoma inhibition assay was performed according to Example 11. Compared with the PBS blank control group, Adc68-Mdpcd1-297 could significantly inhibit tumor growth (Figure 10). In terms of survival time, the death of nude mice in Adc68-Mdpcd1-18 treatment group occurred after 40 days of treatment, however, all control mice died within 40 days. In addition, the average lifespan of the nude mice in the Adc68-Mdpcd1-297 group was significantly prolonged, with about half of the mice surviving more than 58 days. Adc68-Mdpcd1-297 recombinant virus has obvious protective effect on the growth of nude mouse xenograft model.
  • Example 11 for the construction and cell packaging of Ptpcd1-296 recombinant adenovirus.
  • FIG. 11 Example 14. Subcutaneous tumor growth curve of Adc68-Ptpcd1-296 injection-treated and control tumor-bearing mice. **P ⁇ 0.01.
  • the mouse hepatocellular carcinoma tumor inhibition experiment was carried out according to Example 11. Compared with the PBS blank control group, Adc68-Ptpcd1-296 could significantly inhibit the growth of tumor (Figure 11). In terms of survival time, the death of nude mice in Adc68-Mdpcd1-18 treatment group occurred after 40 days of treatment, however, all control mice died within 40 days. In addition, the average lifespan of the nude mice in the Adc68-Ptpcd1-296 group was significantly prolonged, with about half of the mice surviving more than 59 days. Adc68-Ptpcd1-296 recombinant virus has obvious protective effect on the growth of nude mouse xenograft model.
  • nucleotide sequence SEQ ID NO.: 31 of the nucleotide sequence encoding the peptide segment Mdpcd1-307 was synthesized by gene synthesis technology. This sequence encodes 307 amino acids, and the amino acid sequence is shown in SEQ ID NO.:30.
  • the Mdpcd1-307 coding sequence SEQ ID NO.: 31 with both ends connected to the EcoRI and BamHI restriction sites was obtained by gene synthesis, and the gene synthesis product was subjected to the subsequent steps of obtaining the target protein fragment as described in the general method to obtain purification.
  • Example 17 Co-expression with ARF1 enables Mdpcd1-307 protein to obtain destructive test on cells
  • the coding sequence of the above-mentioned protein Mdpcd1-307 and the coding sequence of ARF1 were used to construct transient expression vectors respectively, and the same tobacco leaves were subjected to a 72-hour dip test, and at the same time, they were individually infected as a control.
  • the coding sequence of Mdpcd1-307 was obtained by gene synthesis, and the two ends were connected with the restriction sites of EcoRI and KpnI.
  • the eukaryotic expression vector pEGFP-N1 and the target gene Mdpcd1-307 were digested and purified by EcoRI and KpnI, and the two were connected by T4 DNA ligase.
  • Mdpcd1-303 was directionally cloned into the eukaryotic cell expression vector pEGFP-N1, and then the correctness of the clone was confirmed by enzyme digestion and sequencing.
  • Human hepatoma cell SMMC-7721 was cultured and plated, and when the cell fusion rate was 60%, the cells were grouped for empty vector plasmid and target plasmid transfection, each group had 3 replicate wells, and each group was added with transfection reagent and blue chain-free. Mycin medium. After transfection, the cells were placed in an incubator for 8 hours, and then returned to complete medium containing penicillin-streptomycin after 8 hours, and the cells were harvested for detection after 48 hours. qPCR detection of target gene expression. Flow cytometry analysis of SMMC-7721 cell apoptosis, Mdpcd1-307 can significantly increase the apoptosis of SMMC-7721 cells ( Figure 13).
  • Cell invasion analysis Incubate the chamber covered with Matrigel on the 24-well plate in a cell incubator for more than 6 hours, take it out under sterile conditions, add 100 ⁇ L of pre-warmed serum-free medium to the upper chamber, and let stand at room temperature for 30 hours. After 48 hours of transfection and culture, the cells were digested with 0.25% Trypsin + 0.02% EDTA, centrifuged, resuspended in medium with 2% serum, and counted at 10 ⁇ 10 4 / The density of well is grouped into 24-well plate chambers, and the lower chambers are added with 10% serum medium, 5% CO 2 , and incubated overnight in a 37°C incubator.
  • Mdpcd1-307 recombinant adenovirus construction and cell packaging were completed by commercialization technical services provided by biotechnology companies.
  • mice Nine BABL/c nude mice were randomly divided into blank control group, negative control group and positive intervention group, with 3 mice in each group.
  • a subcutaneous tumorigenic model was established by using SMMC-7721 hepatoma cells at an amount of 1 ⁇ 10 8 /cell.
  • 100 ⁇ L of normal saline was inoculated in the tumor-forming site of the blank control group
  • 100 ⁇ L of 108 U/mL empty vector was inoculated in the negative control group
  • 100 ⁇ L of the corresponding 108 PFU/mL adenovirus was injected in the positive intervention group.
  • the animals in each group were continuously observed, and the changes in food intake and body weight were recorded regularly.
  • Mdpcd1-307 expression effects of Mdpcd1-307 expression on tumor genes in SMMC-7721 liver cancer cells in nude mice, and functional annotation and metabolic pathway enrichment analysis of differentially expressed genes.
  • Mdpcd1-307 expression altered the expression of numerous genes in SMMC-7721 hepatocellular carcinoma cells in nude mice tumors (Fig. 16). These genes were enriched in different metabolic or regulatory pathways associated with tumor development ( Figure 17). Mdpcd1-307 significantly inhibited the tumor development of SMMC-7721 hepatoma cells in nude mice ( Figure 18).
  • Example 20 The destructive test of inhibiting Mdpcd1-303 protein on cells after ARF1 expression interference
  • the Mdpcd1-303 transient expression vector encoding the protein of the above example and the ARF1(LOC103404322)-RNAi expression interference vector were inoculated into the same tobacco leaf, and the ARF1-RNAi expression interference vector was 48 hours earlier than the Mdpcd1-303 transient expression vector Inoculation, the co-infection time was 72 hours, and the individual infection experiments were used as a control.
  • ARF1 expression interference inhibited cell necrosis caused by Mdpcd1-303 overexpression.
  • Example 21 Mdpcd1-303 protein interacts with eEF1a
  • Eukaryotic translation elongation factor eEF1a is highly expressed and plays a key role in tumors (including breast, ovarian and lung cancer, etc.) and many human diseases (Abbas et al., Front.Oncol., 07April 2015
  • Figure 20 is the verification of the interaction between the Mdpcd1-303 protein involved in Example 21 and the yeast two-hybrid protein of eEF1a.
  • Example 22 The destructive test of inhibiting Mdpcd1-303 protein on cells after the interference of eEF1a expression
  • the Mdpcd1-303 transient expression vector encoding the protein of the above example and the eEF1a(LOC103447856)-RNAi expression interference vector were inoculated into the same tobacco leaf, and the eEF1a-RNAi expression interference vector was 48 hours earlier than the Mdpcd1-303 transient expression vector Inoculation, the co-infection time was 72 hours, and the individual infection experiments were used as a control.
  • Example 23 Co-expression with eEF1a enables Mdpcd1-307 protein to obtain destructive test on cells
  • the coding sequence of the above-mentioned protein Mdpcd1-307 and the coding sequence of eEF1a were used to construct transient expression vectors, respectively, and the same tobacco leaf was subjected to a 72-hour dip test, and at the same time, it was separately infected as a control.
  • amino acid sequence with SEQ ID NO.: 1 and/or SEQ ID NO.: 29 can encode the amino acid sequences with SEQ ID NO.: 1 and/or SEQ ID NO.: 29 involved in this paper.
  • the nucleotide sequence of the amino acid sequence shown or the vector including the nucleotide sequence can be used in the application of cell destruction or tumor treatment, in the preparation of compositions for cell destruction, and in the preparation of drugs for tumor treatment.
  • the application of SEQ ID NO.: 1 and/or SEQ ID NO.: 29 can also be prepared or can encode the amino acid sequences with SEQ ID NO.: 1 and/or SEQ ID NO.
  • the nucleotide sequence of the amino acid sequence shown in .:29 or the composition for cell destruction of the vector comprising the nucleotide sequence, or the composition having the nucleotide sequence shown in SEQ ID NO.:1 and/or SEQ ID NO.:29 The amino acid sequence shown or the nucleotide sequence that can encode the amino acid sequence shown in SEQ ID NO.: 1 and/or SEQ ID NO.: 29 involved herein or a vector comprising the nucleotide sequence for use in tumors drug for treatment.
  • ARF1 and eEF1a belong to the same class of GTPases (GTPase), and gene variants in this family of enzymes are associated with the occurrence of various cancers.
  • SEQ ID NO.:1 and/or SEQ ID NO.:29 have the amino acid sequence shown in SEQ ID NO.:1 and/or SEQ ID NO.:29 or can Encoding the nucleotide sequence with the amino acid sequence shown in SEQ ID NO.: 1 and/or SEQ ID NO.: 29 or the vector comprising the nucleotide sequence involved in this paper is used for GTPase gene mutation-related cancers potential drugs for treatment.

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Abstract

本发明提供了一种能破坏细胞的氨基酸序列、编码表达该相应氨基酸序列的核苷酸序列、以及氨基酸序列和核苷酸序列的相关应用，其中，氨基酸序列包括：SEQ ID NO：1和/或SEQ ID NO：29所示的氨基酸序列。

Description

能破坏细胞的氨基酸序列及相关核苷酸序列和相关的应用 技术领域
本发明属于生物技术领域，具体涉及一种能破坏细胞的氨基酸序列、编码该相应氨基酸的核苷酸序列、以及氨基酸序列和核苷酸序列的相关应用。
背景技术
通过一些蛋白表达实现的功能，可以与细胞功能相关的关键蛋白进行互作，以对细胞进行破坏，进而可以用于肿瘤治疗。
例如，ARF1(ADP-ribosylation factor 1)被鉴定为高尔基体分泌运输通路中囊泡形成的关键分子开关(Kahn et al.,The Journal of biological chemistry.1992,267:13039-13046；Beck et al.,The Journal of Cell Biology.2010,194:765–777)，在所有真核生物中，ARF1在其功能和序列特征上都是保守的(Cevher-Keskin,Int.J.Mol.Sci.2013,14,18181-18199)。多项研究报道，ARF1表达与肿瘤细胞复制、增生相关，并被证明为癌症细胞复制、增生的分子开关(Boulay et al.,The Journal of biological chemistry.2008,283:36425-36434；Hashimoto et al.,Proceedings of the National Academy of Sciences of the United States of America.2004,101:6647-6652；Schlienger et al.,Oncotarget,2016,7:11811-11827；Davis et al.,Oncotarget,2016,7:39834-39845)。ARF1被作为相关癌治疗和诊断的关键分子靶点加以研究(Schlienger et al.,Oncotarget,2016,7:11811-11827；Davis et al.,Oncotarget,2016,7:39834-39845；Ohashi et al.,The Journal of Biological Chemistry,2012,287,3885–3897)，化合物AMF-26((2E,4E)-5-((1S,2S,4aR,6R,7S,8S,8aS)-7-hydroxy-2,6,8-trimethyl-1,2,4a,5,6,7,8,8a-octahydronaphthalen-1-yl)-2-methyl-N-(pyridin-3-yl-methyl)penta-2,4-dienamide)通过靶向作用ARF1，诱导高尔基体崩解、细胞坏死及细胞生长抑制，口服AMF-26对人乳腺癌有良好治疗效果(Ohashi et al.2012,J Biol Chem 287(6):3885–3897)。真核细胞翻译延伸因子eEF1α在肿瘤(包括乳腺癌、卵巢癌和肺癌等)和许多人类疾病中高表达并发挥关键作用(Abbas et al.,Front.Oncol.,2015,5:75)。通过水仙环素对eEf1a进行靶向抑制，引起癌细胞凋亡，可以对黑色素瘤进行有效治疗(Van Goietsenoven et al.,FASEB J.2010,24(11):4575-84)。与化学药相比，基因治疗具有在药物制备与治疗中都具有高效率潜能。所以，这时可以通过与ARF1或eEF1a互作，从而启动对细胞的破坏，以达到癌症治疗的效果。
发明内容
本发明提供了一种能破坏细胞的氨基酸序列、编码该相应氨基酸的核苷酸序列、以及氨基酸序列和核苷酸序列的相关应用，以实现对细胞的破坏，为肿瘤治疗提供新的解决方案， 为此，本发明提供了以下的技术方案。
在本发明第一方面，提供一种能破坏细胞的氨基酸序列，所述的破坏细胞指可触发细胞膜系统崩溃，达到破坏细胞的效果，可进一步包括由细胞破坏而引起的组织损伤效果。
本发明提供的氨基酸序列还具有这样的特征：其中，氨基酸序列包括：SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列。
本发明提供的氨基酸序列，还具有这样的特征：其中，氨基酸序列为具有SEQ ID NO.:2所示的氨基酸序列的Mdpcd1-303蛋白片段、或者是将SEQ ID NO.:2的氨基酸残基序列经过一个或一个以上氨基酸残基的取代、缺失或添加，且具有与SEQ ID NO.:2的氨基酸残基序列相同活性的由SEQ ID NO.:2衍生的Mdpcd1-303蛋白片段的衍生蛋白或同源蛋白。
本发明提供的氨基酸序列，还具有这样的特征：其中，衍生蛋白包括Mdpcd1-18蛋白片段和Mdpcd1-297蛋白片段和Mdpcd1-307蛋白片段，同源蛋白包括Ptpcd1-296蛋白片段，Mdpcd1-18蛋白片段具有中SEQ ID NO.:3所示的氨基酸序；Mdpcd1-297蛋白片段具有SEQ ID NO.:4所示的氨基酸序列；Mdpcd1-307蛋白片段具有SEQ ID NO.:30所示的氨基酸序列，Ptpcd1-296蛋白片段具有SEQ ID NO.:5所示的氨基酸序列。
在本发明的第二方面，提供一种核苷酸序列，用于编码得到能破坏细胞的氨基酸序列，其中，氨基酸序列为上述的氨基酸序列。
本发明提供的核苷酸序列，还具有这样的特征：其中，核苷酸序列用于编码Mdpcd1-303蛋白片段、该蛋白片段的衍生蛋白、该蛋白片段的同源蛋白中的任意一种。
本发明提供的核苷酸序列，还具有这样的特征：其中，核苷酸序列用于编码Mdpcd1-303蛋白片段，核苷酸序列为SEQ ID NO.:6所示的核苷酸序列。
本发明提供的核苷酸序列，还具有这样的特征：其中，Mdpcd1-303蛋白片段的衍生蛋白包括Mdpcd1-18蛋白片段和Mdpcd1-297蛋白片段，同源蛋白包括Ptpcd1-296蛋白片段，其中，用于编码Mdpcd1-18蛋白片段的核苷酸序列为SEQ ID NO.:7所示的核苷酸序列，用于编码Mdpcd1-297蛋白片段的核苷酸序列为SEQ ID NO.:8所示的核苷酸序列，用于编码Mdpcd1-307蛋白片段的核苷酸序列为SEQ ID NO.:31所示的核苷酸序列，用于编码Ptpcd1-296蛋白片段的核苷酸序列为SEQ ID NO.:9所示的核苷酸序列。
本发明的第三方面，本发明还提供一种载体(优选地表达载体)，其包括：上述的核苷酸序列。
在本发明的第四方面，提供了所述氨基酸序列，或核苷酸序列，或载体在破坏细胞中的应用，其中，氨基酸序列为上述的氨基酸序列；核苷酸为上述的核苷酸序列；载体为上述的载体。
本发明还提供氨基酸序列，或核苷酸序列，或载体在肿瘤治疗中的应用，其特征在于：其中，氨基酸序列为上述的氨基酸序列；核苷酸为上述的核苷酸序列；载体为上述的载体。
本发明还提供氨基酸序列，或核苷酸序列，或载体在制备用于破坏细胞的组合物中的应用，其特征在于：其中，氨基酸序列为上述的氨基酸序列；核苷酸为上述的核苷酸序列；载体为上述的载体。
本发明还提供氨基酸序列，或核苷酸序列，或载体在制备用于肿瘤治疗的药物中的应用，其特征在于：其中，氨基酸序列为上述的氨基酸序列；核苷酸为上述的核苷酸序列；载体为上述的载体。
在本发明的第五方面，提供一种组合物，其包括：氨基酸序列，或核苷酸序列，或载体，其中，氨基酸序列为上述的氨基酸序列；核苷酸为上述的核苷酸序列；载体为上述的载体。
在另一优选例中，所述组合物还含有药学上可接受的载体。
本发明还提供一种药物组合物，其特征在于，包括：氨基酸序列，或核苷酸序列，或载体，其中，氨基酸序列为上述的氨基酸序列；核苷酸为上述的核苷酸序列；载体为上述的载体。
在本发明的第六方面，提供了破坏细胞的方法，包括步骤：(a)将待破坏的细胞与权利要求1所述的破坏性多肽接触，从而使得所述细胞的细胞膜系统崩溃，从而破坏所述细胞。
在另一优选例中，所述的细胞为哺乳动物细胞。
在另一优选例中，所述的细胞为肿瘤细胞。
在另一优选例中，在步骤(a)中，将表达所述破坏性多肽的核酸或载体导入所述细胞，从而在所述细胞中表达或过表达所述的破坏性多肽。
在另一优选例中，所述方法还包括步骤：(b)检测步骤(a)中所述细胞的细胞膜的完整性和/或细胞是否存活，从而定性或定量地确定所述细胞的破坏情况。
在另一优选例中，所述的方法是非治疗性的和非诊断性的。
在另一优选例中，所述的方法是体外方法。
在另一优选例中，所述的方法是治疗性的。
优选地，本发明获得具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列的Mdpcd1-303及其衍生蛋白或同源蛋白，这些蛋白经证实，可触发细胞膜系统崩溃，达到破坏细胞的效果，并且经证实，编码该序列氨基酸的核苷酸，能对肿瘤具有治疗作用，相应的氨基酸也能对肿瘤具有治疗作用。
利用任何一种可以引导外源基因在肿瘤细胞中适量表达包括超量表达的表达载体，将本发明所提供的蛋白片段Mdpcd1-303或其衍生蛋白片段Mdpcd1-18或其衍生蛋白片段Mdpcd1-297或其衍生蛋白片段Mdpcd1-307或其同源蛋白片段Ptpcd1-296的编码核 苷酸序列导入肿瘤细胞，可改变肿瘤细胞生命进程，启动肿瘤细胞程序性死亡，实现对肿瘤组织的抑制和灭杀，提升机体免疫的作用。使用载体时，其转录起始核苷酸前可加上任何一种增强型启动子或诱导型启动子或肿瘤细胞特异性启动子。含有本发明的上述核苷酸序列的表达载体可通过使用腺病毒、逆转录病毒、腺相关病毒、牛痘病毒、疱疹病毒、慢病毒等各类病毒载体作为基因导入系统转染肿瘤细胞，也可以使用裸质粒DNA、脂质体、阳离子多聚物等方法转染肿瘤细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为实施例9涉及的Mdpcd1-303蛋白片段核苷酸序列瞬时表达载体的浸染试验结果图片。
图2为实施例9涉及的Mdpcd1-18蛋白片段核苷酸序列瞬时表达载体的浸染试验结果图片。
图3为实施例9涉及的Mdpcd1-297蛋白片段核苷酸序列瞬时表达载体的浸染试验结果图片。
图4为实施例9涉及的Ptpcd1-296蛋白片段核苷酸序列瞬时表达载体的浸染试验结果图片。
图5为实施例9涉及的Mdpcd1-303蛋白片段核苷酸序列瞬时表达载体浸染试验后的烟草叶子细胞变化。
图6为实施例9涉及的Atpcd1蛋白核苷酸序列瞬时表达载体的浸染试验结果图片。
图7为实施例10涉及的Mdpcd1-303蛋白与ARF1的酵母双杂交蛋白互作验证(QDO培养基)。
图8为实施例11Adc68-Mdpcd1-303注射处理与对照荷瘤小鼠皮下肿瘤生长曲线。**P<0.01。
图9为实施例12Adc68-Mdpcd1-18注射处理与对照荷瘤小鼠皮下肿瘤生长曲线。**P<0.01。
图10为实施例13Adc68-Mdpcd1-297注射处理与对照荷瘤小鼠皮下肿瘤生长曲线。**P<0.01。
图11为实施例14Adc68-Ptpcd1-296注射处理与对照荷瘤小鼠皮下肿瘤生长曲线。**P<0.01。
图12为实施例17涉及的Mdpcd1-307蛋白片段核苷酸序列瞬时表达载体的浸染试验结果图片。
图13为实施例18涉及的Mdpcd1-307蛋白对SMMC-7721(人肝癌细胞，下同)活力抑制效果的流式细胞仪细胞凋亡检测结果。
图14为实施例18涉及的Mdpcd1-307蛋白对SMMC-7721侵染力抑制检测结果。
图15为实施例19涉及的Mdpcd1-307蛋白促进SMMC-7721肿瘤细胞凋亡率的TUNEL染色检测结果。
图16为实施例19涉及的受Mdpcd1-307蛋白调控SMMC-7721肿瘤差异表达基因数量。
图17为实施例19涉及的受Mdpcd1-307蛋白调控SMMC-7721肿瘤差异表达基因代谢途径功能富集聚类。
图18为实施例19涉及的Mdpcd1-307蛋白对小鼠SMMC-7721肿瘤抑制。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地开发了一种功能独特的源自植物的破坏性多肽，当过表达该破坏性多肽时，可触发细胞膜系统崩溃，达到破坏细胞的效果。具体地，本发明的破坏性多肽Mdpcd1-303源自植物，为植物特有基因，动物中无同源基因；该基因编码的蛋白在过量表达时(如通过35S启动子启动基因表达)可以引起烟草叶片细胞死亡。
此外，本发明的研究还表明，Mdpcd1-303编码蛋白的互作蛋白(例如ARF1或eEF1a)在该基因启动的烟草叶片细胞死亡发挥关键作用；ARF1和eEF1a在动植物中都具有保守性，且与多种肿瘤发生密切相关。
Mdpcd1-307是Mdpcd1-303的等位变异基因，Mdpcd1-307编码的蛋白在过量表达时(由35S启动子启动基因表达)不能引起烟草叶片细胞死亡，但是当Mdpcd1-307与ARF1共表达时，就可以引起烟草叶片细胞死亡；同样，当Mdpcd1-307与eEF1a共表达时，也可以引起烟草叶片细胞死亡。
体外肝癌细胞实验结果显示，Mdpcd1-303可以使肝癌细胞凋亡率显著提升；小鼠活体实验表明，Mdpcd1-303可以显著抑制肿瘤组织生长。Mdpcd1-303的等位基因或核心序列也具有与其类似的功能。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
术语“给药”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内、瘤内、肌内、皮下、腹膜内、脊髓或其它肠胃外给药途径，例如通过注射或输注。
破坏性多肽Mdpcd1-303及其衍生蛋白或同源蛋白
如本文所用，术语“破坏性多肽”、“本发明的多肽”、“本发明的破坏性多肽”可互换使用，指出Mdpcd1-303及其衍生蛋白或同源蛋白。应理解，该术语包括野生型或突变型。此外，该术语还包括全长蛋白或其功能性片段。
一种优选的破坏性蛋白是具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列的Mdpcd1-303及其衍生蛋白(如Mdpcd1-18、Mdpcd1-297、Mdpcd1-307)或同源蛋白(如Ptpcd-296)。
本发明的实验表明，过表达蛋白或大量存在时，可触发细胞膜系统崩溃，达到破坏细胞的效果，并且经证实，编码该序列氨基酸的核苷酸，能对肿瘤具有治疗作用，相应的氨基酸也能对肿瘤具有治疗作用。
此外，利用任何一种可以引导外源基因在肿瘤细胞中适量表达包括超量表达的载体，将本发明所提供的蛋白片段Mdpcd1-303或其衍生蛋白片段Mdpcd1-18或其衍生蛋白片段Mdpcd1-297或其衍生蛋白片段Mdpcd1-307或其同源蛋白片段Ptpcd1-296的编码核苷酸序列导入肿瘤细胞，可改变肿瘤细胞生命进程，启动肿瘤细胞程序性死亡，实现对肿瘤组织的抑制和灭杀，提升机体免疫的作用。
对细胞造成破坏
如本文所用，术语“破坏细胞”或“对细胞造成破坏”可互换使用，指可触发细胞膜系统崩溃，达到破坏细胞的效果，可进一步包括由细胞破坏而引起的组织损伤效果。
在另一优选例中，所述的破坏细胞指对植物细胞、动物细胞、癌细胞等，或其组合，或其对应的组织结构造成破坏或损伤。
在另一优选例中，所述的细胞(包括待被破坏、正在被破坏或已被破坏的细胞)包括(但并不限于)：植物细胞、动物细胞。优选地，为人和非人哺乳动物细胞。
在另一优选例中，所述的细胞为发生病变细胞，如肿瘤细胞。
在另一优选例中，所述的肿瘤细胞包括(但并不限于)：卵巢癌细胞、肺癌细胞、胰腺癌细胞、肝癌细胞、胃癌细胞、乳腺癌细胞、鼻咽癌细胞、食管癌细胞、大肠癌细胞、宫颈癌细胞、白血病及淋巴瘤细胞等。
在另一优选例中，所述的破坏细胞指对烟草叶片细胞及烟草叶片造成破坏与损伤。
在另一优选例中，所述的破坏细胞指对肝癌细胞及肝细胞癌荷瘤造成破坏与损伤。
载体
本发明还提供了含有本发明氨基酸序列或核苷酸序列的载体。源于逆转录病毒诸如腺病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定地整合于细胞基因组中并随子细胞基因组的复制而复制。腺病毒载体转基因效率高，体外实验通常接近100％ 的转导效率；可转导不同类型的人组织细胞，不受靶细胞是否为分裂细胞所限；容易制得高滴度病毒载体；进入细胞内并不整合到宿主细胞基因组，仅瞬间表达，安全性高。因而，腺病毒载体在基因治疗临床试验方面有了越来越多的应用，成为继逆转录病毒载体之后广泛应用且最具前景的病毒载体。
通常，可通过常规操作将本发明的氨基酸序列或核苷酸序列连接至启动子下游，并将其并入表达载体。该载体可整合至真核细胞基因组中并随之复制。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达载体也可用于标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。
所述氨基酸序列或核苷酸序列可被克隆入许多类型的载体。例如，该氨基酸序列或核苷酸序列可被克隆入包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒载体。特定的感兴趣载体包括表达载体、复制载体等。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Molecular Cloning:A Laboratory Manual(Sambrook等，Cold Spring Harbor Laboratory,New York,2001)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺相关病毒、疱疹病毒和慢病毒。通常，合适的载体包含至少一种在有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经有许多基于病毒的系统被开发出来，并用于哺乳动物细胞的基因转导。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒随后可被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些实施方式中，使用腺病毒载体。许多DNA病毒系统在本领域中是已知的。许多腺病毒载体在本领域中是已知的。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些元件位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个元件被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以起动转录。
合适的启动子的一个例子为巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1a(EF-1a)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔病毒(Epstein-Barr virus，EBV)即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸 如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，能够在需要时，启动连接诱导型启动子的多核苷酸序列的表达，或在不需要时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
被引入细胞的表达载体也可包含可选择的标记基因或报告基因中的任一个或两者，以便于通过病毒载体从被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记基因和报告基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记基因包括例如抗生素抗性基因，诸如neomycin等等。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物(如人T细胞)、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、阳离子复合物转染法、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Molecular Cloning:A Laboratory Manual(Sambrook等，Cold Spring Harbor Laboratory,New York,2001)。将多核苷酸引入宿主细胞的优选方法为脂质体法转染法和阳离子复合物聚乙烯亚胺转染法。
将多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。例如见美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂类物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的实施方式中，所述载体为腺病毒载体。
药物组合物以及给药方式
本发明还提供的一种药物组合物为含有任何促进所述能破坏细胞的氨基酸序列和/或核苷酸序列的表达或活性增强的分子实体，或含有所述能破坏细胞的氨基酸序列和/或核苷酸序列，或促进能破坏细胞的氨基酸序列和/或核苷酸序列表达和活性增强的分子实体的表达载体，以及其他药学上可接受的载体。
在本发明的药物组合物，通常含有10 8-10 9PFU的腺病毒颗粒。
如本文所用，术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。它们本身并不是必要的活性成分，且施用后没有过分的毒性。合适的载体是本领域普通技术人员所熟知的。在组合物中药学上可接受的载体可含有液体，如水、盐水、缓冲液。另外，这些载体中还可能存在辅助性的物质，如填充剂、润滑剂、助流剂、润湿剂或乳化剂、pH缓冲物质等。所述的载体中还可以含有细胞转染试剂。
通常，将所述表达载体和药学上可接受的载体混合后，即可获得的本发明的药物组合物。
本发明所述的组合物的给药方式没有特别限制，代表性的例子包括但并不限于：静脉注射、皮下注射、脑部注射、鞘内注射和脊髓注射等。
治疗性应用
本发明所述的含有所述能破坏细胞的氨基酸序列和/或核苷酸序列表达或活性增强的分子实体，或含有所述能破坏细胞的氨基酸序列和/或核苷酸序列，或促进能破坏细胞的氨基酸序列和/或核苷酸序列表达和活性增强的表达载体可用于制备破坏肿瘤细胞，抑制其复制及增生，改变肿瘤细胞的生命进程，启动肿瘤细胞程序性死亡，实现对肿瘤组织的抑制和灭杀，提升机体免疫的药物。且因本发明所述的含有任何促进所述能破坏细胞的氨基酸序列和/或核苷酸序列编码的蛋白与GTP酶类(GTPase)家族(例如ARF1或eEF1a)存在互作关系，而该酶类家族中的基因变异与多种癌症发生相关，故本发明还可用于制备GTPase类基因变异相关癌症治疗的潜在药物。
本发明的主要优点
1.本发明获得了具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列、编码该氨基酸序列的核苷酸序列，以及具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列的Mdpcd1-303及其衍生蛋白或同源蛋白。Mdpcd1-303基因源自植物，为植物特有基因，动物中无同源基因。该基因编码的蛋白在过量表达时(由35S启动子启动基因表达)可以引起细胞死亡，Mdpcd1-303编码蛋白的互作蛋白(例如ARF1或eEF1a)在该基因启动的细胞死亡发挥关键作用。由于ARF1和eEF1a在动植物中都具有保守性，且与多种肿瘤发生密切相关，故本发明所述的具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列、编码该氨 基酸序列的核苷酸序列以及包括该核苷酸序列的载体能通过与动植物体的ARF1和eEF1a互作引发细胞死亡，将植物特有基因的作用范围扩大到了动物之中。
2.Mdpcd1-307是Mdpcd1-303的等位变异基因，Mdpcd1-307编码的蛋白在过量表达时(由35S启动子启动基因表达)不能引起细胞死亡，但是当Mdpcd1-307与ARF1共表达时，就可以引起细胞死亡；同样，当Mdpcd1-307与eEF1a共表达时，也可以引起细胞死亡。进一步说明与ARF1和eEF1a的互作是引发细胞死亡的关键因素，原本不能引起细胞死亡的编码蛋白能通过共表达实现引发细胞死亡的效果。
3.本发明获得具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列的Mdpcd1-303及其衍生蛋白或同源蛋白，这些蛋白经证实，可触发细胞膜系统崩溃，达到破坏细胞的效果。可使肿瘤细胞凋亡率显著提升并显著抑制肿瘤组织生长，从而对肿瘤具有治疗作用。
4.本发明获得的具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列及编码该序列氨基酸的核苷酸也能对肿瘤具有治疗作用。Mdpcd1-303的等位基因或核心序列也具有与其类似的功能。
5.利用任何一种可以引导外源基因在肿瘤细胞中适量表达包括超量表达的载体，将本发明所提供的蛋白片段Mdpcd1-303或其衍生蛋白片段Mdpcd1-18或其衍生蛋白片段Mdpcd1-297或其衍生蛋白片段Mdpcd1-307或其同源蛋白片段Ptpcd1-296的编码核苷酸序列导入肿瘤细胞，即可改变肿瘤细胞生命进程，启动肿瘤细胞程序性死亡，实现对肿瘤组织的抑制和灭杀，提升机体免疫的作用。
6.因本发明所述的含有任何促进所述能破坏细胞的氨基酸序列和/或核苷酸序列编码的蛋白与GTP酶类(GTPase)家族(例如ARF1或eEF1a)存在互作关系，而该酶类家族中的基因变异与多种癌症发生相关，故本发明还可用于制备GTPase类基因变异相关癌症治疗的潜在药物。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。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。除非另外说明，否则百分比和份数是重量百分比和重量份数。
材料与方法
氨基酸序列及核苷酸序列SEQ ID NO.:1(Mdpcd1-9氨基酸序列)
Figure PCTCN2021118063-appb-000001
SEQ ID NO.:2(Mdpcd1-303氨基酸序列)
Figure PCTCN2021118063-appb-000002
SEQ ID NO.:3(Mdpcd1-18氨基酸序列)
Figure PCTCN2021118063-appb-000003
SEQ ID NO.:4(Mdpcd1-297氨基酸序列)
Figure PCTCN2021118063-appb-000004
SEQ ID NO.:5(Ptpcd1-296氨基酸序列)
Figure PCTCN2021118063-appb-000005
SEQ ID NO.:6(Mdpcd1-303核苷酸序列)
Figure PCTCN2021118063-appb-000006
Figure PCTCN2021118063-appb-000007
SEQ ID NO.:7(Mdpcd1-18核苷酸序列)
Figure PCTCN2021118063-appb-000008
SEQ ID NO.:8(Mdpcd1-297核苷酸序列)
Figure PCTCN2021118063-appb-000009
SEQ ID NO.:9(Ptpcd1-296核苷酸序列)
Figure PCTCN2021118063-appb-000010
Figure PCTCN2021118063-appb-000011
SEQ ID NO.:10(P1上游引物核苷酸序列)
Figure PCTCN2021118063-appb-000012
SEQ ID NO.:11(P1下游引物核苷酸序列)
Figure PCTCN2021118063-appb-000013
SEQ ID NO.:12(P2上游引物核苷酸序列)
Figure PCTCN2021118063-appb-000014
SEQ ID NO.:13(P2下游引物核苷酸序列)
Figure PCTCN2021118063-appb-000015
SEQ ID NO.:14(P3上游引物核苷酸序列)
Figure PCTCN2021118063-appb-000016
SEQ ID NO.:15(P3下游引物核苷酸序列)
Figure PCTCN2021118063-appb-000017
SEQ ID NO.:16(P4上游引物核苷酸序列)
Figure PCTCN2021118063-appb-000018
SEQ ID NO.:17(P4下游引物核苷酸序列)
Figure PCTCN2021118063-appb-000019
SEQ ID NO.:18(P5上游引物核苷酸序列)
Figure PCTCN2021118063-appb-000020
SEQ ID NO.:19(P5下游引物核苷酸序列)
Figure PCTCN2021118063-appb-000021
SEQ ID NO.:20(P6上游引物核苷酸序列)
Figure PCTCN2021118063-appb-000022
SEQ ID NO.:21(P6下游引物核苷酸序列)
Figure PCTCN2021118063-appb-000023
SEQ ID NO.:22(P7上游引物核苷酸序列)
Figure PCTCN2021118063-appb-000024
SEQ ID NO.:23(P7下游引物核苷酸序列)
Figure PCTCN2021118063-appb-000025
SEQ ID NO.:24(P8上游引物核苷酸序列)
Figure PCTCN2021118063-appb-000026
SEQ ID NO.:25(P8下游引物核苷酸序列)
Figure PCTCN2021118063-appb-000027
SEQ ID NO.:26(P9上游引物核苷酸序列)
Figure PCTCN2021118063-appb-000028
SEQ ID NO.:27(P9下游引物核苷酸序列)
Figure PCTCN2021118063-appb-000029
SEQ ID NO.:28(Atpcd1氨基酸序列)
Figure PCTCN2021118063-appb-000030
Figure PCTCN2021118063-appb-000031
SEQ ID NO.:29(Mdpcd1-268氨基酸序列)
Figure PCTCN2021118063-appb-000032
SEQ ID NO.:30(Mdpcd1-307氨基酸序列)
Figure PCTCN2021118063-appb-000033
SEQ ID NO.:31(Mdpcd1-307核苷酸序列)
Figure PCTCN2021118063-appb-000034
通用方法
通过PCR扩增获得目的蛋白片段的编码核苷酸序列
参照公布的目的蛋白片段所属的基因组序列，用Macvector软件设计相应的上下游引物。随后用英俊公司的Plant Trizol试剂盒提取相应实验材料的总RNA，用甲醛变性胶电泳鉴定总RNA质量，然后在分光光度计上测定RNA含量。采用Promega的反转录试剂盒进行反转录，合成单链cDNA为模板，用设计的引物扩增目标片段。PCR反应体系为25μL，包含5ng模板，F和R引物各5pmol、2.5μL 10×PCR buffer、37.3nmol MgCl 2、5nmol dNTP、0.5U的rTaq聚合酶。扩增程序为：94℃预变性3min；94℃20s，60℃30s，72℃延伸60s，30个循环；72℃反应5min。
目的蛋白片段的获得
设计引入酶切位点引物，以获得的目的编码核苷酸序列为模板进行PCR扩增，并对PCR产物进行酶切、纯化、定量，克隆到表达载体PET-32a的EcoRⅠ和BamHⅠ酶切位点中，转化E.coli。挑取单菌落于1mL的LB(Amp 100g/mL)中摇菌过夜，转至200mL新鲜的LB培养基中摇至菌液浓度A600≈0.6；加入IPTG至终浓度为1.0mM，37℃培养诱导表达3小时。菌液12,000g离心5min，沉淀悬浮于提取缓冲液中(3M NaCl、1mM PMSF、50mM pH8.0磷酸缓冲液)超声破碎细胞，12,000g离心20min，收集上清。用10mM咪唑、50mM pH8.0磷酸缓冲液平衡Ni-Sepharose凝胶；加入细胞裂解液室温结合20min、用5倍凝胶体积的平衡缓冲液洗涤3次；然后用含300mM咪唑、50mM pH8.0磷酸缓冲液进行洗脱，收集洗脱液即为纯化后的Trx-表达蛋白。纯化后的表达蛋白经透析除盐后按每毫克蛋白样品加入0.1mg肠激酶，于40mM的琥珀酸缓冲液(pH＝5.6)中25℃温育2小时切去组氨酸标签，透析过夜即得到纯化的目的蛋白质片段。
实施例1 Mdpcd1-303蛋白片段的编码核苷酸序列的获得
参照苹果公布的基因组序列，用Macvector软件设计引物P1，P1引物如下：
F-5’-ATGTGTCCAACAAAGCAAAAGC-3’(SEQ ID NO.:10)；
R-5’-TCAATCGTCGTCGTCATCGTCG-3’(SEQ ID NO.:11)。
用英俊公司的Plant Trizol试剂盒提取以苹果(Malus)幼叶为材料的总RNA，采用如通用方法中所述的获得核苷酸序列的后续步骤，通过PCR扩增，获得912bp的核苷酸序列，将PCR产物克隆至pMD18-T载体，测序获得的核苷酸序列如SEQ ID NO.:6所示。该核苷酸序列编码303个氨基酸，该氨基酸序列如SEQ ID NO.:2所示。
实施例2 Mdpcd1-18蛋白片段编码核苷酸序列的获得
通过基因合成技术合成肽段Mdpcd1-18编码核苷酸序列SEQ ID NO.:7。该序列编码18 个氨基酸，该氨基酸序列如SEQ ID NO.:3所示。
实施例3 Mdpcd1-297蛋白片段的编码核苷酸序列的获得
参照苹果公布的基因组序列，用Macvector软件设计引物P2，P2引物如下：
F-5’-ATGTGTCCAACAAAGCAAAAGC-3’(SEQ ID NO.:12)；
R-5’-TCAGCCGGACTTGTGATGATTCAC-3’(SEQ ID NO.:13)。
用英俊公司的Plant Trizol试剂盒提取以苹果(Malus)幼叶为材料的总RNA，采用如通用方法中所述的获得核苷酸序列的后续步骤，通过PCR扩增，获得912bp的核苷酸序列，将PCR产物克隆至pMD18-T载体，测序获得核苷酸序列如SEQ ID NO.:8所示。该核苷酸序列编码297个氨基酸，该氨基酸序列如SEQ ID NO.:4所示。
实施例4 Ptpcd1-296蛋白片段的编码序列的获得
参照毛果杨公布的基因组序列，用Macvector软件设计引物P3，P3引物如下：
F-5’-ATGCACCCAACCAAACAGAA-3’(SEQ ID NO.:14)；
R-5’-TCAATCCTCCTCTTCATCGC-3’(SEQ ID NO.:15)。
用英俊公司的Plant Trizol试剂盒提取以毛果杨(Populus trichocarpa)幼叶为材料的总RNA，采用如通用方法中所述的获得核苷酸序列的后续步骤，通过PCR扩增，获得891bp的核苷酸序列，将PCR产物克隆至pMD18-T载体，测序获得序列如SEQ ID NO.:9所示。该序列编码296个氨基酸，该氨基酸序列如SEQ ID NO.:5所示。
实施例5 蛋白片段Mdpcd1-303的获得
设计引入酶切位点引物P4，P4引物如下：
F-5’-CAGCCCATGGATGTGTCCAACAAAGCAAAAGC-3’(SEQ ID NO.:16)；
R-5’-GAAGTCTAGATCAATCGTCGTCGTCATCGTCG-3’(SEQ ID NO.:17)。
以获得的Mdpcd1-303编码序列SEQ ID NO.:6为模板进行PCR扩增，采用通用方法中所述的获得目的蛋白片段的后续步骤，获得纯化的苹果Mdpcd1的蛋白质片段Mdpcd1-303。
实施例6 蛋白片段Mdpcd1-18的获得
通过基因合成获得两端连接EcoRⅠ和BamHⅠ酶切位点的Mdpcd1-18编码序列SEQ ID NO.:7，并对基因合成产物进行如通用方法中所述的获得目的蛋白片段的后续步骤，获得纯化的蛋白质片段Mdpcd1-18。
鉴于蛋白质片段Mdpcd1-18为小分子肽，还可以通过多肽合成技术直接合成获得。
实施例7 蛋白片段Mdpcd1-297的获得
设计引入酶切位点引物P5，P5引物如下：
F-5’-CAGCCCATGGATGTGTCCAACAAAGCAAAAGC-3’(SEQ ID NO.:18)；
R-5’-GAAGTCTAGATCAGCCGGACTTGTGATGATTCAC-3’(SEQ ID NO.:19)。
以获得的Mdpcd1核苷酸序列SEQ ID NO.:8为模板进行PCR扩增，采用通用方法中所述的获得目的蛋白片段的后续步骤，获得纯化的蛋白质片段Mdpcd1-297。
实施例8 蛋白片段Ptpcd1-296的获得
设计引入酶切位点引物P6，P6引物如下：
F-5’-CAGCCCATGGATGCACCCAACCAAACAGAA-3’(SEQ ID NO.:20)；
R-5’-GAAGTCTAGATCAATCCTCCTCTTCATCG-3’(SEQ ID NO.:21)。
以获得的Mdpcd核苷酸1序列SEQ ID NO.:9为模板进行PCR扩增，采用通用方法中所述的获得目的蛋白片段的后续步骤，获得纯化的蛋白质片段Ptpcd1-296。
对于上述获得的各个蛋白，其中，SEQ ID NO.:1和/或SEQ ID NO.:29为保守功能区。
实施例9 获得的各个蛋白对细胞的破坏性试验
本实施例中，将上述各个实施例蛋白的编码序列构建瞬时表达载体，分别对烟草叶子进行72小时浸染试验，同时做空载体对照。
另外，还采用与Mdpcd1-303蛋白同源的拟南芥Atpcd1蛋白编码序列瞬时表达载体进行同样的浸染试验，Atpcd1蛋白氨基酸序列如SEQ ID NO.:28所示，该序列没有上述各个实施例获得的蛋白的保守序列：SEQ ID NO.:1和/或SEQ ID NO.:29。
图1为实施例9涉及的Mdpcd1-303蛋白片段编码序列瞬时表达载体的浸染试验结果图片；
图2为实施例9涉及的Mdpcd1-18蛋白片段编码序列瞬时表达载体的浸染试验结果图片；
图3为实施例9涉及的Mdpcd1-297蛋白片段编码序列瞬时表达载体的浸染试验结果图片；
图4为实施例9涉及的Ptpcd1-296蛋白片段编码序列瞬时表达载体的浸染试验结果图片。
如图1-4所示，经过浸染试验，相对空载对照来说，浸染后，对烟草叶片造成了损伤。
图5为实施例9涉及的Mdpcd1-303蛋白片段编码序列瞬时表达载体浸染试验后的烟草叶片细胞的变化。
如图5所示，将Mdpcd1-303蛋白片段编码序列瞬时表达载体浸染试验后遭到损伤的烟草叶片(A区域)置于细胞视野中，定位于细胞膜的Mdpcd1-303-GFP融合蛋白伴随细胞膜破裂而弥散分解(图5中，GF：GFP荧光位置即为细胞膜位置，GFP荧光信号即Mdpcd1-303的定位信息；BF：白光下的细胞膜解体图像；Merged：即BF与GF叠加后的重叠吻合情况，可表明Mdpcd1-303-GFP定位于细胞膜，且伴随细胞膜破裂而弥散分解)，说明Mdpcd1-303 蛋白片段对细胞具有破坏性。根据图2-5，还推测其衍生或同源片段也对细胞具有破坏性。
图6为实施例9涉及的Atpcd1蛋白编码序列瞬时表达载体的浸染试验结果图片。
如图6所示，Atpcd1蛋白编码序列瞬时表达载体浸染后，烟草叶片无坏死现象，所以进一步证实，氨基酸序列SEQ ID NO.:1和/或SEQ ID NO.:29为造成细胞破坏的保守功能区。由于氨基酸序列SEQ ID NO.:1和/或SEQ ID NO.:29具有生物普遍性，所以可以推测包括该序列的氨基酸序列也能对动物细胞造成破坏。
实施例10 蛋白片段Mdpcd1-303与ARF1酵母双杂交蛋白互作分析
图7为实施例10涉及的Mdpcd1-303蛋白与ARF1的酵母双杂交蛋白互作验证(QDO培养基)。
构建Mdpcd1诱饵质粒、AFR1猎物质粒重组质粒，对构建的诱饵质粒和猎物质粒大量抽提并进行琼脂糖凝胶电泳检测。ARF1蛋白具有跨物种的序列和功能保守性，实验中使用的苹果ARF1序列(LOC103404322)来自公共数据库，共用共知。将获得的诱饵质粒进行毒性检测及自激活检测，将诱饵质粒和猎物空载共转化NMY51感受态，涂布DDO平板能生长说明重组诱饵质粒成功转入宿主菌且对宿主菌无毒性，涂布TDO和QDO不能生长，说明诱饵蛋白不能激活报告基因表达。将诱饵质粒和质粒POST-NubaI共转化NMY51感受态，涂布平板DDO能生长，共转化成功，涂布TDO和QDO均有菌落生长，说明激活了报告基因HIS，ADE2，说明bait构建的读码框正确，ubiquitin实验系统可行使功能。将猎物质粒与诱饵质粒共转化酵母细胞，对照和功能验证结果符合预期，说明该系统可用于双杂交验证；猎物质粒与诱饵质粒转化的宿主菌，涂布DDO能生长，说明涂布SD-TLHA能生长，说明二者之间存在相互作用(图7)。
本实施例说明，具有氨基酸序列SEQ ID NO.:1和/或SEQ ID NO.:29的蛋白片段可能会通过与ARF1之间存在着的互作，对细胞造成破坏。
实施例11 蛋白质片段Mdpcd1-303的编码核苷酸序列的应用
以Mdpcd1-303重组腺病毒对肝细胞癌荷瘤鼠动物模型的治疗为例。
鉴于Mdpcd303作用靶点ARF1在动、植物中的基础性和保守性，因此Mdpcd1-303等具有氨基酸序列SEQ ID NO.:1和/或SEQ ID NO.:29的蛋白片段介导的细胞程序性死亡在动、植物中具有保守性，这些蛋白介导的肿瘤等病变细胞增殖抑制、消杀和死亡具有广泛性特征。本实施例为众多类似具有氨基酸序列SEQ ID NO.:1和/或SEQ ID NO.:29的Mdpcd1-303蛋白片段功能应用实施之一。
Mdpcd1-303重组腺病毒构建和细胞包装由生物技术公司提供的商业化技术服务完成。
转移质粒pShuttle-CMV-Mdpcd1-303的构建：
PCR扩增Mdpcd1-cDNA模板，Mdpcd1-303上游引物P7：
5′-AGCCACCATGGATGTGTCCAACAAAGCAAAAGC-3′(SEQ ID NO.:22)；
下游引物：
5′-GTACCTCTAGATCAATCGTCGTCGTCATCGTCG-3′(SEQ ID NO.:23)。
预期扩增片段940bp，PCR反应条件为：94℃3min；94℃20s，58℃30s，72℃1min，30个循环；72℃7min。1％琼脂糖胶电泳分离后切胶回收。转移质粒pShuttle-CMV及目的基因Mdpcd1经KpnⅠ，XbaⅠ酶切纯化，由T4DNA连接酶连接两者，将Mdpcd1-303定向克隆入转移质粒pShuttle-CMV中，然后酶切及测序鉴定克隆的正确性。重组腺病毒的构建及病毒在HEK293细胞中的包装，扩增，纯化及鉴定：鉴定后确认克隆成功的转移质粒pShutttle-CMV-Mdpcd1-303及骨架质粒pAdc68经PI-sceⅠ及IceuⅠ酶切线性化，电泳鉴定正确后，切胶回收，16℃连接过夜，氯化钙法转化E.coli stab-2，铺LB平板(氨苄霉素)筛选，挑克隆，摇菌，质粒抽提，质粒经BaglⅡ，XholⅠ，MunⅠ酶切及测序正确。重组阳性克隆pShuttle-CMV-Mdpcd1-303-pAdc68构建成功。HEK293细胞生长至约80％融合时，pShuttle-CMV-Mdpcd1-303-pAdc68经PacⅠ酶切线性化，采用X-treme法转染HEK293细胞，使病毒在HEK293细胞中包装，培养12d，光镜下见细胞变圆，从培养瓶底壁脱落，细胞核占据大部分细胞体积，即发生细胞病理效应(cytopathiceffect,CPE)，收集细胞，3,500r/min离心5min，去上清加无抗无血清DMEM重悬，37℃、-80℃反复冻融3次，再次感染HEK293细胞扩增，反复扩增3～4代后CsCl梯度离心纯化，获得带有Mdpcd1-303基因的腺病毒载体(命名为Adc68-Mdpcd1-303)，-80℃保存备用。
图8为实施例11Adc68-Mdpcd1-303注射处理与对照荷瘤小鼠皮下肿瘤生长曲线。**P<0.01。
随机抽取30只肝细胞癌成瘤实验成功的小鼠，并随机分为2组，分别接受瘤内注射：实验组Adc68-Mdpcd1-303(1×10 9PFU)；空白对照组100μL PBS；每隔3天测量小鼠的肿瘤体积，随时观察小鼠成活情况。肿瘤体积测量公式为体积＝长度/2×宽度 2。与PBS空白对照组相比较，Adc68-Mdpcd1-303可明显抑制肿瘤的生长(图8)。在生存时间上，Adc68-Mdpcd1-303治疗组裸鼠死亡均发生于治疗40天之后，然而，所有的对照组小鼠在40天内死亡。此外，Adc68-Mdpcd1-303组裸鼠的平均寿命延长显著，其中约半数小鼠存活超过55天。Adc68-Mdpcd1-303重组病毒对裸鼠移植瘤模型的生长具有明显保护效应。
实施例12 蛋白质片段Mdpcd1-18的编码序列的应用：
以Mdpcd1-18重组腺病毒对肝细胞癌荷瘤鼠动物模型的治疗为例。
Mdpcd1-18重组腺病毒构建和细胞包装按照实施例11，人工合成Mdpcd1-18的编码核苷酸序列，合成时在编码核苷酸序列两端加入KpnⅠ，XbaⅠ酶切位点。
图9为实施例12Adc68-Mdpcd1-18注射处理与对照荷瘤小鼠皮下肿瘤生长曲线。**P<0.01。
小鼠肝细胞癌瘤抑制实验参照实施例11。与PBS空白对照组相比较，Adc68-Mdpcd1-18可明显抑制肿瘤的生长(图9)。在生存时间上，Adc68-Mdpcd1-18治疗组裸鼠死亡均发生于 治疗40天之后，然而，所有的对照组小鼠在40天内死亡。此外，Adc68-Mdpcd1-18组裸鼠的平均寿命延长显著，其中约半数小鼠存活超过60天。Adc68-Mdpcd1-18重组病毒对裸鼠移植瘤模型的生长具有明显保护效应。
实施例13 蛋白质片段Mdpcd1-297的编码序列的应用
以Mdpcd1-297重组腺病毒对肝细胞癌荷瘤鼠动物模型的治疗为例。
Mdpcd1-297重组腺病毒构建和细胞包装参照实施例11。
转移质粒pShuttle-CMV-Mdpcd1-297构建用PCR引物P8：
上游引物：
5′-AGCCACCATGGATGTGTCCAACAAAGCAAAAGC-3′(SEQ ID NO.:24)；
下游引物：
5′-GTACCTCTAGATCAGCCGGACTTGTGATGAT-3′(SEQ ID NO.:25)。
图10为实施例13Adc68-Mdpcd1-297注射处理与对照荷瘤小鼠皮下肿瘤生长曲线。**P<0.01。
小鼠肝细胞癌瘤抑制实验按照按照实施例11。与PBS空白对照组相比较，Adc68-Mdpcd1-297可明显抑制肿瘤的生长(图10)。在生存时间上，Adc68-Mdpcd1-18治疗组裸鼠死亡均发生于治疗40天之后，然而，所有的对照组小鼠在40天内死亡。此外，Adc68-Mdpcd1-297组裸鼠的平均寿命延长显著，其中约半数小鼠存活超过58天。Adc68-Mdpcd1-297重组病毒对裸鼠移植瘤模型的生长具有明显保护效应。
实施例14 蛋白质片段Ptpcd1-296的编码序列的应用
以Ptpcd1-296重组腺病毒对肝细胞癌荷瘤鼠动物模型的治疗为例。
Ptpcd1-296重组腺病毒构建和细胞包装参照实施例11。
转移质粒pShuttle-CMV-Ptpcd1-296构建用PCR引物P9：
上游引物：
5′-AGCCACCATGCACCCAACCAAACAGAAACAC-3′(SEQ ID NO.:26)；
下游引物：
5′-GTACCTCTAGATCAATCCTCCTCTTCATCGC-3′(SEQ ID NO.:27)。
图11实施例14Adc68-Ptpcd1-296注射处理与对照荷瘤小鼠皮下肿瘤生长曲线。**P<0.01。
小鼠肝细胞癌瘤抑制实验按照实施例11。与PBS空白对照组相比较，Adc68-Ptpcd1-296可明显抑制肿瘤的生长(图11)。在生存时间上，Adc68-Mdpcd1-18治疗组裸鼠死亡均发生于治疗40天之后，然而，所有的对照组小鼠在40天内死亡。此外，Adc68-Ptpcd1-296组裸鼠的平均寿命延长显著，其中约半数小鼠存活超过59天。Adc68-Ptpcd1-296重组病毒对裸鼠移植瘤模型的生长具有明显保护效应。
实施例15 Mdpcd1-307蛋白片段的编码序列的获得
通过基因合成技术合成肽段Mdpcd1-307编码核苷酸序列SEQ ID NO.:31。该序列编码307个氨基酸，该氨基酸序列如SEQ ID NO.:30所示。
实施例16 Mdpcd1-307蛋白片段的获得
通过基因合成获得两端连接EcoRⅠ和BamHⅠ酶切位点的Mdpcd1-307编码序列SEQ ID NO.:31，并对基因合成产物进行如通用方法中所述的获得目的蛋白片段的后续步骤，获得纯化的蛋白质片段Mdpcd1-307，其中，SEQ ID NO.:1和/或SEQ ID NO.:29为保守功能区。
实施例17 与ARF1共表达使Mdpcd1-307蛋白获得对细胞的破坏性试验
本实施例中，将上述实施例蛋白Mdpcd1-307的编码序列与ARF1(LOC103404322)编码序列分别构建瞬时表达载体，对同一烟草叶子进行72小时浸染试验，同时分别单独侵染做为对照。
如图12所示，ARF1与Mdpcd1-307超量表达时启动细胞坏死。
实施例18 蛋白质片段Mdpcd1-307的编码核苷酸序列的应用
以Mdpcd1-307重组质粒转染对SMMC-7721细胞活力的抑制为例。
通过基因合成获得两端连接EcoRⅠ和KpnⅠ酶切位点的Mdpcd1-307编码序列，真核细胞表达载体pEGFP-N1及目的基因Mdpcd1-307经EcoRⅠ和KpnⅠ酶切纯化，由T4DNA连接酶连接两者，将Mdpcd1-303定向克隆入真核细胞表达载体pEGFP-N1中，然后酶切及测序鉴定克隆的正确性。将人的肝癌细胞SMMC-7721进行培养、铺板，待细胞融合率60％时分组进行空载质粒、目的质粒转染，每个组3个复孔，每组加入转染试剂以及不含青链霉素的培养基。转染后，放在培养箱中干扰8h，8h后换回含青链霉素完全培养基，48h后收取细胞做检测。qPCR检验目的基因表达。流式细胞仪检测分析SMMC-7721细胞凋亡，Mdpcd1-307能使SMMC-7721细胞凋亡显著增加(图13)。细胞侵袭分析：将24孔板上室内铺好基质胶的小室在细胞培养箱中孵育6h以上，在无菌条件下取出，上室中加入100μL预温的无血清培养基，室温下静置30分钟，使基质胶水化，再吸去剩余培养基；转染培养48h后细胞，用0.25％Trypsin+0.02％EDTA消化离心，用2％血清的培养基重悬，计数，以10×10 4/well的密度分组铺24孔板上室，下室加入10％血清的培养基，5％CO 2，37℃培养箱孵育过夜培养。12h培养后，1×PBS清洗3次，用4％多聚甲醛室温固定15min，再用1×PBS清洗3次，用棉签擦去上室细胞，加入结晶紫染色15min，用1×PBS清洗3次，置于室温下风干，显微镜下拍照。实验重复三次。Mdpcd1-307能使SMMC-7721细胞侵袭能力显著下降(图14)。
实施例19 蛋白质片段Mdpcd1-307的编码核苷酸序列的应用
以Mdpcd1-307对接种SMMC-7721肝癌细胞裸鼠的抗肿瘤效果为例。
Mdpcd1-307重组腺病毒构建和细胞包装由生物技术公司提供的商业化技术服务完成。
转移质粒pShuttle-CMV-Mdpcd1-307的构建参照实施例11。
将9只BABL/c裸鼠随机分为空白对照组，阴性对照组，阳性干预组，每组3只。SMMC-7721肝癌细胞1×10 8/只的量构建皮下成瘤模型。接种癌细胞第3d后，空白对照组成瘤部位接种100μL生理盐水，阴性对照组接种108U/mL空载体100μL，阳性干预组注射相应的108PFU/mL腺病毒100μL。干预结束后连续观察各组动物，定时记录的摄食量及体重变化。待可见成瘤后，每5d记录荷瘤鼠生长情况及皮下移植瘤大小，用于绘制生长曲线。于接种后20d终止实验，固定样品备用。按照常规实验流程进行肿瘤组织切片制备和Tunel染色，结果显示，Mdpcd1-307的表达使SMMC-7721肝癌细胞裸鼠肿瘤细胞凋亡率显著高于空白对照组或阴性对照组(图15)。参照标准分子实验流程，提取各个样品RNA，进行文库构建和高通量测序，对测序数据进行质量控制、参考基因锚定和基因表达丰度计算，以空白对照组或阴性对照组为参照，分析Mdpcd1-307表达对SMMC-7721肝癌细胞裸鼠肿瘤基因的影响，对差异表达基因进行功能注释和代谢途径富集分析。相对于空白对照组或阴性对照组，Mdpcd1-307表达改变SMMC-7721肝癌细胞裸鼠肿瘤中众多基因的表达(图16)。这些基因富集于肿瘤发育相关的不同代谢或调控途径(图17)。Mdpcd1-307对SMMC-7721肝癌细胞裸鼠肿瘤发育存在明显抑制作用(图18)。
实施例20 ARF1表达干扰后抑制Mdpcd1-303蛋白对细胞的破坏性试验
本实施例中，将上述实施例蛋白的编码序列Mdpcd1-303瞬时表达载体与ARF1(LOC103404322)-RNAi表达干扰载体接种同一烟草叶子，ARF1-RNAi表达干扰载体早于Mdpcd1-303瞬时表达载体48小时接种，共同浸染时间72小时，同时以分别单独侵染实验做为对照。
如图19所示，ARF1表达干扰后抑制了Mdpcd1-303超量表达引起的细胞坏死。
实施例21 Mdpcd1-303蛋白与eEF1a互作
真核细胞翻译延伸因子eEF1a在肿瘤(包括乳腺癌、卵巢癌和肺癌等)和许多人类疾病中高表达并发挥关键作用(Abbas et al.,Front.Oncol.,07April 2015|https://doi.org/10.3389/fonc.2015.00075)。所以，这时可以将eEF1a作为靶标，通过与eEF1a互作，启动对细胞的破坏，以达到癌症等疾病治疗的效果。
图20为实施例21涉及的Mdpcd1-303蛋白与eEF1a的酵母双杂交蛋白互作验证。
实施例22 eEF1a表达干扰后抑制Mdpcd1-303蛋白对细胞的破坏性试验
本实施例中，将上述实施例蛋白的编码序列Mdpcd1-303瞬时表达载体与 eEF1a(LOC103447856)-RNAi表达干扰载体接种同一烟草叶子，eEF1a-RNAi表达干扰载体早于Mdpcd1-303瞬时表达载体48小时接种，共同浸染时间72小时，同时以分别单独侵染实验做为对照。
如图21所示，eEF1a表达干扰后抑制了Mdpcd1-303超量表达引起的细胞坏死。
实施例23 与eEF1a共表达使Mdpcd1-307蛋白获得对细胞的破坏性试验
本实施例中，将上述实施例蛋白Mdpcd1-307的编码序列与eEF1a(LOC103447856)编码序列分别构建瞬时表达载体，对同一烟草叶子进行72小时浸染试验，同时分别单独侵染做为对照。
如图22所示，eEF1a与Mdpcd1-307超量表达时启动细胞坏死。
讨论
上述实施例说明，具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列或能编码本文所涉及的具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列的核苷酸序列或包括该核苷酸序列的载体，可以实现在细胞破坏或肿瘤治疗的应用、在制备细胞破坏的组合物中的应用、在制备用于肿瘤治疗的药物中的应用，从而也能制备得到包括具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列或能编码本文所涉及的具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列的核苷酸序列或包括该核苷酸序列的载体的用于细胞破坏的组合物、或包括具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列或能编码本文所涉及的具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列的核苷酸序列或包括该核苷酸序列的载体的用于肿瘤治疗的药物。ARF1与eEF1a同属于GTP酶类(GTPase)，该酶类家族中的基因变异与多种癌症发生相关，具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列或能编码本文所涉及的具有SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列的核苷酸序列或包括该核苷酸序列的载体，是用于GTPase类基因变异相关癌症治疗的潜在药物。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (20)

  1. 一种能破坏细胞的氨基酸序列，其特征在于，所述的破坏细胞指可触发细胞膜系统崩溃，达到破坏细胞的效果，可进一步包括由细胞破坏而引起的组织损伤效果。
  2. 如权利要求1所述的能破坏细胞的氨基酸序列，其特征在于，所述的氨基酸序列选自SEQ ID NO.:1和/或SEQ ID NO.:29所示的氨基酸序列。
  3. 如权利要求2所述的氨基酸序列，其特征在于，所述氨基酸序列选自下组：
    (1)具有SEQ ID NO.:2所示的氨基酸序列的Mdpcd1-303蛋白片段；
    (2)将SEQ ID NO.:2的氨基酸残基序列经过一个或一个以上氨基酸残基的取代、缺失或添加，且具有与SEQ ID NO.:2的氨基酸残基序列相同活性的由SEQ ID NO.:2衍生的所述Mdpcd1-303蛋白片段的衍生蛋白或同源蛋白。
  4. 如权利要求3所述的氨基酸序列，其特征在于，所述的衍生蛋白选自下组：Mdpcd1-18蛋白片段、Mdpcd1-297蛋白片段、Mdpcd1-307蛋白片段，或其组合。
    所述同源蛋白包括Ptpcd1-296蛋白片段，
    其中，所述Mdpcd1-18蛋白片段具有SEQ ID NO.:3所示的氨基酸序列；
    所述Mdpcd1-297蛋白片段具有SEQ ID NO.:4所示的氨基酸序列；
    所述Ptpcd1-296蛋白片段具有SEQ ID NO.:5所示的氨基酸序列；
    所述Mdpcd1-307蛋白片段具有SEQ ID NO.:30所示的氨基酸序列。
  5. 一种能破坏细胞的核苷酸序列，其特征在于，所述的核苷酸序列用于编码得到能破坏细胞的氨基酸序列，其中所述氨基酸序列为权利要求2-4任意一项所述的氨基酸序列。
  6. 如权利要求5所述的核苷酸序列，其特征在于，所述的核苷酸序列用于编码Mdpcd1-303蛋白片段、该蛋白片段的衍生蛋白、该蛋白片段的同源蛋白，或其组合。
  7. 如权利要求6所述的核苷酸序列，其特征在于，所述的核苷酸序列用于编码Mdpcd1-303蛋白片段，所述核苷酸序列具有SEQ ID NO.:6所示的核苷酸序列。
  8. 如权利要求5所述的核苷酸序列，其特征在于，所述的Mdpcd1-303蛋白片段的衍生蛋白包括Mdpcd1-18蛋白片段、Mdpcd1-297蛋白片段和Mdpcd1-307蛋白片段，所述同源蛋白包括Ptpcd1-296蛋白片段，
    其中，用于编码所述Mdpcd1-18蛋白片段的核苷酸序列为SEQ ID NO.:7所示的核苷酸序列，
    用于编码所述Mdpcd1-297蛋白片段的核苷酸序列为SEQ ID NO.:8所示的核苷酸序列，
    用于编码所述Ptpcd1-296蛋白片段的核苷酸序列为SEQ ID NO.:9所示的核苷酸序列,
    用于编码所述Mdpcd1-307蛋白片段的核苷酸序列为SEQ ID NO.:31所示的核苷酸序列。
  9. 一种载体，其特征在于，包含权利要求5-8任意一项所述的核苷酸序列。
  10. 氨基酸序列，或核苷酸序列，或载体在破坏细胞中的应用，其特征在于，所述氨基 酸序列为权利要求2-4任意一项所述的氨基酸序列；所述核苷酸为权利要求5-8任意一项所述的核苷酸序列；所述载体为权利要求9所述的载体。
  11. 氨基酸序列，或核苷酸序列，或载体在肿瘤治疗中的应用，其特征在于，所述氨基酸序列为权利要求2-4任意一项所述的氨基酸序列；所述核苷酸为权利要求5-8任意一项所述的核苷酸序列；所述载体为权利要求8所述的载体。
  12. 氨基酸序列，或核苷酸序列，或载体在制备用于破坏细胞的组合物中的应用，其特征在于，所述氨基酸序列为权利要求2-4任意一项所述的氨基酸序列；所述核苷酸为权利要求5-8任意一项所述的核苷酸序列；所述载体为权利要求9所述的载体。
  13. 氨基酸序列，或核苷酸序列，或载体在制备用于肿瘤治疗的药物中的应用，其特征在于，所述氨基酸序列为权利要求2-4任意一项所述的氨基酸序列；所述核苷酸为权利要求5-8任意一项所述的核苷酸序列；所述载体为权利要求9所述的载体。
  14. 一种用于细胞破坏的组合物，其特征在于，包括：氨基酸序列，或核苷酸序列，或载体，其中，所述氨基酸序列为权利要求2-4任意一项所述的氨基酸序列；所述核苷酸为权利要求5-8任意一项所述的核苷酸序列；所述载体为权利要求9所述的载体。
  15. 一种用于肿瘤治疗的药物组合物，其特征在于，包括：
    氨基酸序列，或核苷酸序列，或载体，其中，所述氨基酸序列为权利要求2-4任意一项所述的氨基酸序列；所述核苷酸为权利要求5-8任意一项所述的核苷酸序列；所述载体为权利要求9所述的载体。
  16. 一种体外的破坏细胞的方法，其特征在于，包括步骤：(a)将待破坏的细胞与权利要求1所述的破坏性多肽接触，从而使得所述细胞的细胞膜系统崩溃，从而破坏所述细胞。
  17. 如权利要求16所述的方法，其特征在于，所述的细胞为哺乳动物细胞。
  18. 如权利要求16所述的方法，其特征在于，所述的细胞为肿瘤细胞。
  19. 如权利要求16所述的方法，其特征在于，在步骤(a)中，将表达所述破坏性多肽的核酸或载体导入所述细胞，从而在所述细胞中表达或过表达所述的破坏性多肽。
  20. 如权利要求16所述的方法，其特征在于，所述方法还包括步骤：(b)检测步骤(a)中所述细胞的细胞膜的完整性和/或细胞是否存活，从而定性或定量地确定所述细胞的破坏情况。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1164829A (zh) * 1994-09-08 1997-11-12 光固化有限公司 将分子导入细胞溶质
CN1836729A (zh) * 2006-04-24 2006-09-27 杭州埃夫朗生化制品有限公司 用于杀伤肿瘤细胞的靶向性抗体复合物及其制备方法
CN102174097A (zh) * 2011-01-25 2011-09-07 中国人民解放军第二军医大学 一种海葵溶细胞素及其应用
CN105670998A (zh) * 2016-01-15 2016-06-15 浙江大学 一种钙化癌细胞的方法
CN109069624A (zh) * 2016-01-15 2018-12-21 瑞美控股有限责任公司 癌症的免疫治疗
CN110747155A (zh) * 2019-11-12 2020-02-04 湖南大学 减毒重组工程菌及其制备方法、应用和肿瘤靶向药物

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114163508B (zh) * 2020-09-11 2024-07-16 王月志 能破坏细胞的氨基酸序列及相关核苷酸序列和相关的应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1164829A (zh) * 1994-09-08 1997-11-12 光固化有限公司 将分子导入细胞溶质
CN1836729A (zh) * 2006-04-24 2006-09-27 杭州埃夫朗生化制品有限公司 用于杀伤肿瘤细胞的靶向性抗体复合物及其制备方法
CN102174097A (zh) * 2011-01-25 2011-09-07 中国人民解放军第二军医大学 一种海葵溶细胞素及其应用
CN105670998A (zh) * 2016-01-15 2016-06-15 浙江大学 一种钙化癌细胞的方法
CN109069624A (zh) * 2016-01-15 2018-12-21 瑞美控股有限责任公司 癌症的免疫治疗
CN110747155A (zh) * 2019-11-12 2020-02-04 湖南大学 减毒重组工程菌及其制备方法、应用和肿瘤靶向药物

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