Composition for inhibiting tumor cells as well as preparation and application thereof

Abstract

The invention discloses a composition for inhibiting tumor cells as well as preparation and application thereof. The composition comprises a protein or nucleic acid of Escherichia coli cytosine deaminase (CD), and a protein or nucleic acid of transmembrane protein CD63. The transmembrane protein CD63 of the exosome is utilized to autonomously wrap the protein and RNA of the suicide gene CD into the exosome, so that the drug loading rate is increased. The growth of human non-small cell lung cancer cells is successfully inhibited by detecting the proliferation condition of target cells after the 5-Fc drug is added.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a composition for inhibiting tumor cells and its preparation and application. Background Art

[0002] Suicide gene therapy offers a new strategy for cancer treatment. Cells expressing a suicide gene are killed upon exposure to a specific drug. The Escherichia coli cytosine deaminase (CD) gene is one such target. It binds to 5-fluorocytosine (5-Fc) and catalyzes the hydrolytic deamination of the non-toxic 5-Fc molecule into 5-fluorouracil (5-FU). This 5-FU is then converted intracellularly into other cytotoxic metabolites that incorporate into DNA and RNA, leading to cell cycle arrest and apoptosis. Studies have shown that the active triphosphate form of 5-FU can freely diffuse across the cell membrane, exerting a potent bystander effect (killing not only the transgenic cells but also the large number of surrounding non-transgenic cells). However, this approach is limited in its therapeutic efficacy due to its lack of specificity and low delivery efficiency.

[0003] Exosomes are membrane-enclosed extracellular vesicles (EVs) with diameters ranging from 40 nm to 160 nm, with an average size of about 100 nm. They originate from endosomes, and the invagination of the cytoplasmic membrane can form multivesicular bodies. Multivesicular bodies fuse with other intracellular vesicles and organelles, providing different types of raw materials for the formation of exosomes.

[0004] Exosomes contain a rich array of transmembrane proteins, cell adhesion molecules, scaffold proteins, RNA-binding proteins, RNA, DNA, and complex glycans, but the enrichment of these substances varies among different exosomes. Differences in exosome composition, particularly differences in cell surface proteins, can have varying effects on recipient cells. Specific cell surface markers also enable the purification of specific exosome populations. Studies have found that four transmembrane proteins (CD81, CD82, CD37, and CD63) are highly enriched in exosomes and play a crucial role in the transport, stabilization, and oligomerization of related membrane proteins.

[0005] In recent years, research has confirmed the therapeutic potential of exosomes for a wide range of diseases. For example, studies have found that exosomes can cross the blood-brain barrier and act as regulators of inflammatory responses and neural regeneration. Exosomes isolated from cardiosphere-derived cells, when injected into a mouse model of ischemia, can inhibit apoptosis and induce cardiac cell growth. Furthermore, exosomes offer numerous advantages over other drug delivery vehicles, including good biocompatibility, low immunogenicity, the ability to avoid clearance by macrophages, thereby prolonging drug retention, and the ability to cross the blood-brain barrier for brain delivery.

[0006] Therefore, using exosomes as a drug delivery method has become a potential effective way to diagnose and treat diseases, and the prospects are bright. Although the use of exosomes as drug or gene delivery carriers is still in its infancy, with the deepening of exosome research, exosome therapy may eventually lead to major breakthroughs in the field of drug or gene delivery. Summary of the Invention

[0007] To address the shortcomings of existing suicide gene therapy approaches, one objective of the present invention is to provide a drug delivery method that uses exosomes as drug carriers and the suicide gene CD as cargo to deliver it to target cells. A second objective of the present invention is to utilize exosome membrane proteins to autonomously encapsulate the suicide gene CD protein and RNA into exosomes, thereby increasing drug loading efficiency.

[0008] In one aspect, the present invention provides a composition comprising one or more of the following:

[0009] 1) Escherichia coli cytosine deaminase CD and transmembrane protein CD63;

[0010] 2) nucleic acid encoding Escherichia coli cytosine deaminase CD and transmembrane protein CD63;

[0011] 3) Escherichia coli cytosine deaminase CD and nucleic acid encoding transmembrane protein CD63;

[0012] 4) Nucleic acid encoding Escherichia coli cytosine deaminase CD and nucleic acid encoding transmembrane protein CD63;

[0013] 5) a fusion protein of Escherichia coli cytosine deaminase CD and transmembrane protein CD63; and,

[0014] 6) Nucleic acid encoding the fusion protein of 5).

[0015] In some embodiments, the composition satisfies one or more of the following conditions:

[0016] 1) The Escherichia coli cytosine deaminase CD has the amino acid sequence shown in SEQ ID NO: 9;

[0017] 2) The transmembrane protein CD63 has the amino acid sequence shown in SEQ ID NO: 10;

[0018] 3) the nucleic acid encoding Escherichia coli cytosine deaminase CD has the sequence shown in SEQ ID NO: 1; and,

[0019] 4) The nucleic acid encoding the transmembrane protein CD63 has a sequence as shown in SEQ ID NO: 2 or 11.

[0020] Escherichia coli cytosine deaminase CD and transmembrane protein CD63 ultimately function in the form of proteins. For the Escherichia coli cytosine deaminase CD protein, its nucleic acid (RNA) can be first transferred into the body and then translated into the Escherichia coli cytosine deaminase CD protein in the body.

[0021] In some embodiments, the composition further comprises 5-fluorocytosine and / or ganciclovir.

[0022] The CD gene is often used in combination with the herpes simplex virus thymidine kinase (HSV-TK) gene to form a dual suicide gene system. HSV-TK converts ganciclovir (GCV) to the toxic GCV triphosphate, which in turn inhibits DNA synthesis and produces cytotoxic effects. This dual suicide gene system can enhance anti-tumor efficacy because the two suicide gene systems can function simultaneously, increasing tumor cell sensitivity to the drug. This system can simultaneously utilize two prodrugs (5-FC and GCV) to produce cytotoxicity through different metabolic pathways, thereby enhancing anti-tumor efficacy.

[0023] Another aspect of the present invention provides a recombinant expression vector or a recombinant expression vector composition, wherein the recombinant expression vector or the recombinant expression vector composition satisfies any one of the following conditions:

[0024] 1) The recombinant expression vector carries a nucleic acid encoding Escherichia coli cytosine deaminase CD and a nucleic acid encoding transmembrane protein CD63;

[0025] 2) the recombinant expression vector carries a nucleic acid encoding a fusion protein of Escherichia coli cytosine deaminase CD and transmembrane protein CD63; and,

[0026] 3) The recombinant expression vector composition comprises: an expression vector carrying a nucleic acid encoding Escherichia coli cytosine deaminase CD, and an expression vector carrying a nucleic acid encoding a transmembrane protein CD63.

[0027] In some embodiments, the nucleic acid encoding Escherichia coli cytosine deaminase CD has a sequence as shown in SEQ ID NO: 1; and / or the nucleic acid encoding transmembrane protein CD63 has a sequence as shown in SEQ ID NO: 2 or 11.

[0028] In some embodiments, the recombinant expression vector is a plasmid or viral expression vector.

[0029] In some embodiments, the recombinant expression vector or recombinant expression vector composition satisfies one or both of the following conditions:

[0030] 1) The nucleic acid encoding Escherichia coli cytosine deaminase CD is a ribonucleic acid and contains a C / D box stem-loop structure; and,

[0031] 2) The expression vector carrying the nucleic acid encoding the transmembrane protein CD63 also contains a nucleic acid sequence encoding the L7Ae protein.

[0032] In some specific embodiments, the backbone of the lentiviral expression vector is a pLVX series vector or a pCDH series vector; for example, the pLVX series vector is pLVX-EF1α-IRES-puro.

[0033] Another aspect of the present invention provides a transformant, which is a host cell comprising the recombinant expression vector or recombinant expression vector composition provided by the present invention, and the transformant is a non-animal or plant species.

[0034] In some embodiments, the host cell is a mammalian cell, such as a stem cell, an immune cell, a tumor cell, an ovarian cell, or a kidney cell.

[0035] In some embodiments, the tumor cells are, for example, engineered B16-F10 cells or MCF7 cells.

[0036] In some embodiments, the kidney cells are selected from 293 cells, such as 293T cells; and the ovarian cells are selected from CHO cells.

[0037] Another aspect of the present invention provides an exosome, wherein the exosome comprises the composition provided by the present invention or is secreted by the transformant provided by the present invention.

[0038] In some embodiments, the exosomes contain Escherichia coli cytosine deaminase CD and transmembrane protein CD63, and the transmembrane protein can be native to the exosomes or introduced exogenously.

[0039] In some embodiments, the exosomes contain nucleic acid encoding Escherichia coli cytosine deaminase CD and transmembrane protein CD63, and the transmembrane protein can be native to the exosomes or introduced exogenously.

[0040] Another aspect of the present invention provides a pharmaceutical composition, comprising the composition provided by the present invention, the recombinant expression vector or recombinant expression vector composition provided by the present invention, the transformant provided by the present invention, or the exosome provided by the present invention.

[0041] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0042] Another aspect of the present invention provides a method for preparing the exosomes provided by the present invention, the method comprising culturing the transformant provided by the present invention and obtaining exosomes from the culture.

[0043] Another aspect of the present invention provides a method for increasing the enrichment of Escherichia coli cytosine deaminase CD in exosomes, the method comprising combining a nucleotide encoding Escherichia coli cytosine deaminase CD or a CD protein with a transmembrane protein CD63.

[0044] In some embodiments, the method comprises conjugating nucleotides encoding E. coli cytosine deaminase CD to the transmembrane protein CD63.

[0045] In some embodiments, the method comprises combining a protein encoding E. coli cytosine deaminase CD with the transmembrane protein CD63.

[0046] In some embodiments, the recombinant expression vector or recombinant expression vector composition provided by the present invention, or the transformant provided by the present invention is used.

[0047] In some embodiments, the enrichment level of Escherichia coli cytosine deaminase CD in exosomes is increased by preparing the recombinant expression vector or recombinant expression vector composition provided by the present invention, or the transformant provided by the present invention.

[0048] In some embodiments, the method comprises the following steps:

[0049] A recombinant expression vector or a recombinant expression vector composition is prepared, the recombinant expression vector or the recombinant expression vector composition is transferred into mammalian cell culture, and exosomes are collected after culture; the recombinant expression vector or the recombinant expression vector composition is the recombinant expression vector or the recombinant expression vector composition defined in the present invention.

[0050] Another aspect of the present invention provides a method for inhibiting tumor cells, comprising contacting tumor cells with the composition provided by the present invention, the exosomes provided by the present invention, or the pharmaceutical composition provided by the present invention; the method is for non-diagnostic treatment purposes.

[0051] In some embodiments, the tumor cell is non-small cell lung cancer A549 cell.

[0052] Another aspect of the present invention provides use of the composition, recombinant expression vector or recombinant expression vector composition, transformant, exosome or pharmaceutical composition provided by the present invention in the preparation of a product for a cell proliferative disorder.

[0053] In some embodiments, the cell proliferative disorder is selected from at least one of non-small cell lung cancer, lung cancer, colon cancer, gastric cancer, and pancreatic cancer.

[0054] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0055] The reagents and raw materials used in the present invention are commercially available.

[0056] The positive advances of this invention include providing a method for delivering CD suicide gene protein and RNA using exosome membrane proteins. By monitoring the proliferation of target cells after the addition of 5-Fc, the growth of human non-small cell lung cancer cells was successfully inhibited. Therefore, this invention provides an effective method for gene therapy using CD suicide gene protein and RNA via the exosome pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is the construction map of the pLVX-EF1a-CD63-CD-IRES-Puro-mRFP plasmid.

[0058] Figure 2 This figure shows the effect of 5-Fc drug on the proliferation of transfected 293T cells detected by CCK8 after transient transfection of the plasmid in 293T cells.

[0059] Figure 3 This is the result of using Western Blot to detect marker proteins in CELL (cells) and EXO (exosomes).

[0060] Figure 4 After A549 cells were incubated with EXO (CTRL), EXO (CD) and EXO (CD63-CD), the expression of CD protein in A549 cells was detected by Western Blot.

[0061] Figure 5 This is a graph using CCK8 to detect the effect of 5-Fc drug on the proliferation of transfected A549 cells. The upper part is protein delivery and the lower part is RNA delivery.

[0062] Figure 6 This is the construction map of the pLVX-EF1a-CD63-L7Ae-IRES-Puro plasmid.

[0063] Figure 7 This is the construction map of the pLVX-EF1a-Myc-CD-C / Dbox-IRES-Puro-mRFP plasmid.

[0064] Figure 8 The results of qPCR detection of RNA content in exo-293T and exo-293TCD.

[0065] Figure 9 This is the construction map of the pLVX-EF1a-EGFP-C / Dbox-IRES-Puro plasmid. DETAILED DESCRIPTION

[0066] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0067] CELL refers to the cell pellet after collecting the supernatant.

[0068] CD81 is one of the exosome marker proteins.

[0069] EXO is the abbreviation of exosome, which stands for exosome.

[0070] TSG101: present in both cells and exosomes, used together with other marker proteins to identify cells and exosomes.

[0071] Calcnexin: It only exists in cells and should not exist in exosomes. It is used to distinguish exosome components from cellular components.

[0072] Serum-free culture medium was purchased from Wuhan Punosai Life Science Co., Ltd.; fetal bovine serum (FBS) was purchased from Shanghai Bosheng Biotechnology Co., Ltd.; and double antibody (penicillin-streptomycin mixture) was purchased from Beijing Solaibao Technology Co., Ltd.

[0073] Example 1 Construction of exosome plasmids for delivering CD gene protein and RNA via the exosome membrane protein pathway

[0074] 1. Amplification of membrane protein and CD gene fusion expression sequence

[0075] The GeneBank database (https: / / www.ncbi.nlm.nih.gov / ) was queried for the sequence information of the CD (cytosinedeaminase, codA, AY331712.1) gene (the nucleotide sequence of CD is shown in SEQ ID NO: 1) and CD63 (NM_001780) (the nucleotide sequence of CD63 is shown in SEQ ID NO: 2). Amplification primers were designed. After amplification, primers were designed using the overlapping PCR principle to overlap the CD gene and CD63, and a Myc tag protein was added between the two. The primer sequences are shown in Table 1, and the reaction procedure is shown in Table 2:

[0076] Table 1. Sequence amplification primer sequences and related information

[0077]

[0078] Table 2. Amplification reaction program

[0079]

[0080] 2. Amplification of membrane protein and L7Ae fusion expression sequence

[0081] After gene synthesis of the L7Ae sequence, overlapping PCR primers were designed to bridge the L7Ae sequence to the CD63 protein. The primer sequences are shown in Table 3, and the reaction procedure is shown in Table 2.

[0082] Table 3. Sequence amplification primer sequences and related information

[0083]

[0084] 3. Vector linearization

[0085] (1) Get the target fragment

[0086] Use pLVX-EF1a-IRES-Puro (purchased from Miaoling Plasmid) to add mRFP to form a new lentiviral expression vector pLVX-EF1a-IRES-Puro-mRFP. The new lentiviral expression vector is double-digested with EcoRI / BamHI to linearize the vector. The enzyme digestion system is shown in Table 4:

[0087] Table 4. Enzyme digestion system

[0088]

[0089] The enzyme digestion reaction was carried out at 37°C for 3 h, and the vector digestion products were subjected to 1% agarose gel electrophoresis to recover the target fragment.

[0090] (2) Ligation of target fragment and vector

[0091] The linearized pLVX-EF1a-IRES-Puro-mRFP obtained in step (1) was homologously recombined with the PCR product obtained in step (1). The homologous recombination system is shown in Table 5:

[0092] Table 5. Homologous recombination system

[0093]

[0094] The reaction was incubated at 37°C for 30 min before the conversion experiment.

[0095] (3) PCR identification and sequencing of transformation and positive clones

[0096] Add 5 μL of the ligation product obtained in step (2) to 50 μL of E. coli competent cells, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, and ice bath for 2 minutes; add 500 μL of LB liquid culture medium without antibiotics, shake and culture at 37°C for 1 hour at 200 rpm; take the bacterial liquid and evenly spread it on the LB solid culture medium containing Amp, and culture it in a 37°C incubator overnight. The next day, pick a single colony, perform bacterial liquid PCR identification, select positive clones for sequencing (Suzhou Jinweizhi Biotechnology Co., Ltd.), and after sequencing comparison, identify the positive clones and obtain the successfully constructed plasmid:

[0097] Protein group: pLVX-EF1a-CD63-IRES-Puro-mRFP plasmid, pLVX-EF1a- Myc-CD-IRES-Puro-mRFP plasmid, pLVX-EF1a-CD63-Myc-CD-IRES-Puro-mRFP plasmid;

[0098] RNA group: pLVX-EF1a-L7Ae-IRES-Puro-mRFP plasmid and pLVX-EF1a-Myc-C / Dbox-IRES-Puro-mRFP plasmid;

[0099] pLVX-EF1a-CD63-L7Ae-IRES-Puro-mRFP plasmid and pLVX-EF1a-Myc-C / Dbox-IRES-Puro-mRFP plasmid

[0100] pLVX-EF1a-L7Ae-IRES-Puro-mRFP plasmid and pLVX-EF1a-Myc-CD-C / Dbox-IRES-Puro-mRFP plasmid (CD RNA control group);

[0101] pLVX-EF1a-CD63-L7Ae-IRES-Puro-mRFP plasmid and pLVX-EF1a-Myc-CD-C / Dbox-IRES-Puro-mRFP plasmid (gene synthesis), plasmids as Figure 1 、 Figure 6 and Figure 7 、 Figure 9 shown.

[0102] 4. Plasmid Verification

[0103] (1) Cell culture

[0104] Human renal epithelial cell line 293T was cultured in DMEM supplemented with 10% fetal bovine serum and 1% PBS at 37°C in an incubator with 5% CO2 and 90% relative humidity. Cells were routinely digested using 0.25% trypsin containing EDTA and plated at a density of 70% to 80% in six-well plates.

[0105] (2) Cell transfection

[0106] Experimental groups:

[0107] Blank control group: pLVX-EF1a-IRES-Puro-mRFP (blank control group);

[0108] Experimental control group: pLVX-EF1a-CD-IRES-Puro-mRFP (CD control group);

[0109] Experimental group: pLVX-EF1a-CD63-Myc-CD-IRES-Puro-mRFP (CD63-CD group);

[0110] pLVX-EF1a-Myc-CD-C / Dbox-IRES-Puro-mRFP (CD-C / D RNA group);

[0111] The day after plating, prepare the transfection complex: For each well of cells, dilute 2 μg of target plasmid DNA in 200 μL of serum-free medium. Mix thoroughly to make a DNA dilution solution. Immediately add 4 μL of PEI transfection reagent and mix gently. Incubate at room temperature for 15 minutes to allow the DNA-PEI nucleic acid transfection reagent complex to form.

[0112] During complex formation, remove the cell growth medium and add 1 mL of pre-warmed complete culture medium (containing 10% fetal bovine serum and 1% double-antibody) to each well. Add 200 μL of DNA-PEI complex directly to the cells and gently shake the culture plate to mix.

[0113] After 6 hours, the culture medium containing the DNA-PEI complex was removed, and 2 mL of pre-warmed complete culture medium was added to each well to continue culturing.

[0114] (3) Proliferation assay

[0115] 48 h after transfection, the cells of the transfection control group and the experimental group were digested with trypsin, centrifuged and resuspended in complete culture medium, and the cell concentration was adjusted to 3×10 3 Each well was inoculated in a 96-well plate, 100 μL / well, and 3 replicates were set up for each group.

[0116] After the cells adhered, the cell proliferation was detected at 0 h using the CCK8 detection kit (Dojindo, Japan). At the same time, 100 μL of complete culture medium containing 1 mM 5-Fc was added to each well. The culture was continued for 96 h, and the cell proliferation was detected every 24 h. The results are shown in the figure. Figure 2 As shown, the cell proliferation of the CD63-CD group was inhibited by about 60% compared with the CTRL group after the addition of 5-Fc.

[0117] Example 2 Exosome collection, extraction and identification

[0118] 1. Cell and transfection preparation

[0119] Experimental groups:

[0120] Blank control group: pLVX-EF1a-IRES-Puro-mRFP (blank control group)

[0121] CD control group: pLVX-EF1a-Myc-CD-IRES-Puro-mRFP (CD control group)

[0122] Protein delivery experimental group: pLVX-EF1a-CD63-Myc-CD-IRES-Puro-mRFP (CD63-CD group)

[0123] RNA delivery control group: pLVX-EF1a-L7Ae-IRES-Puro-GFP + pLVX-EF1a-Myc-C / Dbox-IRES-Puro-mRFP (RNA blank control group)

[0124] RNA delivery experimental group: pLVX-EF1a-L7Ae-IRES-Puro-GFP + pLVX-EF1a-Myc-CD-C / Dbox-IRES-Puro-mRFP (CD RNA control group)

[0125] RNA delivery experimental group: pLVX-EF1a-CD63-L7Ae-IRES-Puro-GFP + pLVX-EF1a-Myc-CD-C / Dbox-IRES-Puro-mRFP (CD63+CD RNA group)

[0126] 293T cells were passaged to 70-80% confluency and transfected with PEI according to experimental groups. After 24 hours, the supernatant was discarded and the cells were washed twice with PBS to remove the serum-containing medium. A certain volume of serum-free medium was added for culture. After 48-72 hours, the cell supernatant was collected.

[0127] 2. Ultrafiltration method to separate exosomes

[0128] The collected supernatant was centrifuged at 4°C, 2000g for 30 min, and the supernatant was collected; a second round of centrifugation was performed at 4°C, 4000g, and the supernatant was collected; a third round of centrifugation was performed at 4°C, 12000g, and the supernatant was collected. After three rounds of centrifugation, the supernatant was filtered through a 0.22μm membrane and the filtrate was collected.

[0129] Use PBS to balance a 100KD ultrafiltration tube and centrifuge at 4000g until all PBS is filtered out; add the filtrate to the ultrafiltration tube, centrifuge repeatedly at 4000g, and concentrate to about 500μL; add PBS to the concentrate in the ultrafiltration tube for buffer exchange, and continue ultrafiltration at 4°C and 4000g centrifugation to concentrate to less than 200μL. At this time, the exosomes contain more than 100KD of miscellaneous proteins.

[0130] 3. Detection of exosome proteins

[0131] (1) Protein extraction

[0132] Add lysis buffer to the exosome pellet and add PMSF when needed. Mix thoroughly and centrifuge at 12,000 rpm for 20-30 min at 4°C to collect the supernatant.

[0133] Determine protein concentration using a BCA kit. Dilute the protein appropriately so that the measured concentration is within the linear range of the standard curve. Use the BCA kit to determine protein concentration in triplicate. The final original protein concentration should be above 1 mg / mL. Quickly freeze protein above 100 μg in liquid nitrogen and store at -80°C.

[0134] (2) Western blotting of exosome proteins

[0135] The protein after BSA quantification was subjected to Western Blot detection. Figure 3 , CD81, Calnexin (calcium-binding protein), and TSG101 indicate the exosomes extracted by the present invention, and Myc is a tag antibody of the CD gene, indicating the expression of the CD gene. Figure 3 The results showed that the blank control group did not express CD protein in the cells, while both the CD control group and the CD63-CD group expressed CD protein; in the exosomes, the blank control group did not express CD protein, while both the CD control group and the CD63-CD group expressed CD protein, and the amount of CD protein expressed in the CD63-CD group was significantly higher than that in the CD control group, indicating that the CD63-CD group achieved the enrichment of CD protein.

[0136] Detection of exosomal RNA

[0137] (1) RNA extraction

[0138] Exosome RNA was extracted using an RNA rapid extraction kit.

[0139] (2) Fluorescence quantitative PCR detection

[0140] The extracted RNA was reverse transcribed into cDNA (Conway Reverse Transcription Kit), and qPCR primers and SYBR Green were used for on-chip detection. The results were as follows: Figure 8 , CD-RNA group and CD63-CD RNA were successfully encapsulated in exosomes.

[0141] Example 3 Exosome delivery experiment

[0142] 1. Cell incubation

[0143] (1) Recipient cells: A549

[0144] Human non-small cell lung cancer A549 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum and 1% PBS at 37°C in a 5% CO2 incubator with a relative humidity of 90%. They were routinely digested using 0.25% trypsin containing EDTA and plated at a density of 60% to 70% in 12-well plates.

[0145] (2) Equal amounts of exosomes from the blank control group (pLVX-EF1a-IRES-Puro-mRFP), CD control group (pLVX-EF1a-Myc-CD-IRES-Puro-mRFP), and experimental CD63-CD group (pLVX-EF1a-CD63-Myc-CD-IRES-Puro-mRFP) were taken and filtered with a 0.22 μm membrane. The mixture was mixed with complete culture medium (1640 culture medium, 10% fetal bovine serum, 1% double antibody) and added to a 12-well plate for further culture.

[0146] 2. Detection of exosome-delivered proteins

[0147] (1) Protein extraction

[0148] After cell culture, centrifuge to obtain the cell pellet, add lysis buffer to the cell pellet, add PMSF when needed, mix thoroughly, centrifuge at 12000 rpm at 4°C for 20-30 minutes, and collect the supernatant.

[0149] (2) Western blot detection

[0150] The protein after BSA quantification was detected by western blot. The results were as follows Figure 4 , Actin is the internal reference protein, and Myc is the tag antibody of CD gene, indicating the expression of CD gene. Figure 4The results showed that the expression of CD protein could be detected after incubation of A549 cells in the experimental group, while it was not detected in the control group, indicating that CD63 can successfully deliver CD protein to the target cells.

[0151] Example 4 CD protein and RNA delivered via exosomal membrane proteins in combination with 5-Fc inhibited the proliferation of human non-small cell lung cancer cells

[0152] (1) Experimental groups:

[0153] Blank control group: pLVX-EF1a-IRES-Puro-mRFP (blank control group)

[0154] Protein delivery control group: pLVX-EF1a-Myc-CD-IRES-Puro-mRFP (CD control group);

[0155] Protein delivery experimental group: pLVX-EF1a-CD63-Myc-CD-IRES-Puro-mRFP (CD63-CD group);

[0156] RNA delivery control group: pLVX-EF1a-L7Ae-IRES-Puro-GFP + pLVX-EF1a-Myc-C / Dbox-IRES-Puro-mRFP (RNA blank control group);

[0157] RNA delivery experimental group: pLVX-EF1a-L7Ae-IRES-Puro-GFP + pLVX-EF1a-Myc-CD-C / Dbox-IRES-Puro-mRFP (CD RNA control group)

[0158] RNA delivery experimental group: pLVX-EF1a-CD63-L7Ae-IRES-Puro-GFP + pLVX-EF1a-Myc-CD-C / Dbox-IRES-Puro-mRFP (CD63+CD RNA group);

[0159] (2) Take human non-small cell lung cancer A549 cells in the logarithmic growth phase and adjust the cell concentration to 4×10 3 Each well was inoculated in a 96-well plate, with 3 replicates per group.

[0160] (3) After the cells adhered, the CCK8 detection kit was used to detect the cell proliferation at 0 h. At the same time, 100 μL of complete culture medium was added to each well. In the protein delivery experiment group, the blank control group, CD control group, and CD63-CD group were added or not added with 1 mM 5-Fc, respectively. In the RNA delivery experiment group, the RNA blank control group, CD RNA control group, and CD63+CDRNA group were added or not added with 1 mM 5-Fc.

[0161] (4) Continue culturing for 96 hours and monitor cell proliferation every 24 hours. The results are as follows: Figure 5 From the perspective of protein delivery, when 5-Fc was not added, there was little difference between the blank control group, CD control group, and CD63-CD group, indicating that CD and CD63 themselves had little effect on cell proliferation. When 5-Fc was added, the CD63-CD group was more able to inhibit cell proliferation than the blank control group and CD control group, with the CD63-CD group inhibiting approximately 73% and the CD control group inhibiting approximately 59%, while the blank control group was not significantly inhibited. From the perspective of RNA delivery, when 5-Fc was not added, there was little difference between the RNA blank control group, CDRNA control group, and CD63-CD RNA group, indicating that CD and CD63 themselves had little effect on cell proliferation. When 5-Fc was added, the CD63-CD RNA group was more able to inhibit cell proliferation than the RNA blank control group and CD RNA control group, with the CD63-CD RNA group inhibiting approximately 74% and the CD RNA control group inhibiting approximately 44%, while the blank control group was not significantly inhibited. This proves that the CD gene is enriched in exosomes through the membrane protein CD63, and can be successfully delivered to target cells in the form of RNA and protein, respectively, and react with 5-Fc, thereby inhibiting cell growth.

[0162] The nucleotide sequence of CD is shown in SEQ ID NO: 1

[0163]

[0164] The nucleotide sequence of CD63 is shown in SEQ ID NO: 2

[0165] ATGGCGGTGGAAGGAGGAATGAAATGTGTGAAGTTCTTGCTCTACGTCCTCCTGCTGGCCTTTTGCGCCTGTGCAGTGGGACTGATTGCCGTGGGTGTCGGGGCACAGCTTGTCCTGAGTCAGACCATAATCCAGGGGGCTACCCCTGGCTCTCTGTTGCCAGTGGTCATCATCGCAGTGGGTGTCTTCCTCTTCCTGGTGGCTTTTGTGGGCTGCTGCGGGGCCTGCAAGGAGAACTATTGTCTTATGATCACGTTTGCCATCTTTCTGTCTCTTATCATGTTGGTGGAGGTGGCCGCAGCCATTGCTGGCTATGTGTTTAGAGATAAGGTGATGTCAGAGTTTAATAACAACTTCCGGCAGCAGATGGAGAATTACCCGAAAAACAACCACACTGCTTCGATCCTGGACAGGATGCAGGCAGATTTTAAGTGCTGTGGGGCTGCTAACTACACAGATTGGGAGAAAATCCCTTCCATGTCGAAGAACCGAGTCCCCGACTCCTGCTGCATTAATGTTACTGTGGGCTGTGGGATTAATTTCAACGAGAAGGCGATCCATAAGGAGGGCTGTGTGGAGAAGATTGGGGGCTGGCTGAGGAAAAATGTGCTGGTGGTAGCTGCAGCAGCCCTTGGAATTGCTTTTGTCGAGGTTTTGGGAATTGTCTTTGCCTGCTGCCTCGTGAAGAGTATCAGAAGTGGCTACGAGGTGATGTAG

[0166] The amino acid sequence of CD is shown in SEQ ID NO: 9

[0167] MSNNALQTIINARLPGEEGLWQIHLQDGKISAIDAQSGVMPITENSLDAEQGLVIPPFVEHIHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVKQRAWQTLKWQIANGIQHVRTHVDVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSYPNGEALLEEALRLGADVVGAIPHFEFTREYGVESLHKTFALAQKYDRLIDVHCDEIDDEQSRFVETVAALAHREGMGARVTASHTTAMHSYNGAYTSRLFRLLKMSGINFVANPLVNIHLQGRFDTYPKRRGITRVKEMLESGINVCFGHDDVFDPWYPLGTANMLQVLHMGLHVCQLMGYGQINDGLNLITHHSARTLNLQDYGIAAGNSANLIILPAENGFDALRRQVPVRYSVRGGKVIASTQPAQTTVYLEQPEAIDYKR

[0168] The amino acid sequence of CD63 is shown in SEQ ID NO: 10

[0169] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM

[0170] The nucleotide sequence of CD63 is shown in SEQ ID NO: 11

[0171] AUGGCGGUGGAAGGAGGAAUGAAAUGUGUGAAGUUCUUGCUCUACGUCCUCCUGCUGGCCUUUUGCGCCUGUGCAGUGGGACUGAUUGCCGUGGGUGUCGGGGCACAGCUUGUCCUGAGUCAGACCAUAAUCCAGGGGGCUACCCCUGGCUCUCUGUUGCCAGUGGUCAUCAUCGCAGU GGGUGUCUUCCUCUUCCUGGUGGCUUUUGUGGGCUGCUGCGGGGCCUGCAAGGAGAACUAUUGUCUUAUGAUCACGUUUGCCAUCUUUCUGUCUCUUAUCAUGUUGGUGGAGGUGGCCGCAGCCAUUGCUGGCUAUGUGUUUAGAGAUAAGGUGAUGUCAGAGUUUAAUAACAACUUCC GGCAGCAGAUGGAGAAUUACCCGAAAAACAACCACACUGCUUCGAUCCUGGACAGGAUGCAGGCAGAUUUUAAGUGCUGUGGGGCUGCUAACUACACAGAUUGGGAGAAAAUCCCUUCCAUGUCGAAGAACCGAGUCCCCGACUCCUGCUGCAUUAAUGUUACUGUGGGCUGUGGGAUU AAUUUCAACGAGAAGGCGAUCCAUAAGGAGGGCUGUGUGGAGAAGAUUGGGGGCUGGCUGAGGAAAAAUGUGCUGGUGGUAGCUGCAGCAGCCCUUGGAAUUGCUUUUGUCGAGGUUUUGGGAAUUGUCUUUGCCUGCUGCCUCGUGAAGAGUAUCAGAAGUGGCUACGAGGUGAUGUAG

[0172] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A composition, characterized in that The composition comprises one or more of the following: 1) Escherichia coli cytosine deaminase CD and transmembrane protein CD63; 2) nucleic acid encoding Escherichia coli cytosine deaminase CD and transmembrane protein CD63; 3) Escherichia coli cytosine deaminase CD and nucleic acid encoding transmembrane protein CD63; 4) Nucleic acid encoding Escherichia coli cytosine deaminase CD and nucleic acid encoding transmembrane protein CD63; 5) Fusion protein of Escherichia coli cytosine deaminase CD and transmembrane protein CD63; and, 6) Nucleic acid encoding the fusion protein of 5).

2. The composition according to claim 1, wherein The composition satisfies one or more of the following conditions: 1) The Escherichia coli cytosine deaminase CD has the amino acid sequence shown in SEQ ID NO: 9; 2) The transmembrane protein CD63 has the amino acid sequence shown in SEQ ID NO: 10; 3) the nucleic acid encoding Escherichia coli cytosine deaminase CD has the sequence shown in SEQ ID NO: 1; and, 4) The nucleic acid encoding the transmembrane protein CD63 has a sequence as shown in SEQ ID NO: 2 or 11; Preferably, the composition further comprises 5-fluorocytosine and / or ganciclovir.

3. A recombinant expression vector or a recombinant expression vector composition, characterized in that: The recombinant expression vector or recombinant expression vector composition meets any of the following conditions: 1) The recombinant expression vector carries a nucleic acid encoding Escherichia coli cytosine deaminase CD and a nucleic acid encoding transmembrane protein CD63; 2) the recombinant expression vector carries a nucleic acid encoding a fusion protein of Escherichia coli cytosine deaminase CD and transmembrane protein CD63; and, 3) The recombinant expression vector composition comprises: an expression vector carrying a nucleic acid encoding Escherichia coli cytosine deaminase CD, and an expression vector carrying a nucleic acid encoding transmembrane protein CD63; Preferably, the nucleic acid encoding Escherichia coli cytosine deaminase CD has a sequence as shown in SEQ ID NO: 1; and / or the nucleic acid encoding transmembrane protein CD63 has a sequence as shown in SEQ ID NO: 2 or 11.

4. The recombinant expression vector or recombinant expression vector composition according to claim 3, wherein: The recombinant expression vector is a plasmid or viral expression vector; Preferably, the recombinant expression vector or recombinant expression vector composition satisfies one or both of the following conditions: 1) The nucleic acid encoding Escherichia coli cytosine deaminase CD is a ribonucleic acid and contains a C / D box stem-loop structure; and, 2) The expression vector carrying the nucleic acid encoding the transmembrane protein CD63 also contains a nucleic acid sequence encoding the L7Ae protein; More preferably, the backbone of the viral expression vector is a pLVX series vector or a pCDH series vector; for example, the pLVX series vector is pLVX-EF1α-IRES-puro.

5. A transformant, characterized in that: The transformant is a host cell comprising the recombinant expression vector or the recombinant expression vector composition according to claim 3 or 4; the transformant is a non-animal or plant species; Preferably, the host cell is a mammalian cell, such as a stem cell, an immune cell, a tumor cell, an ovarian cell or a kidney cell; More preferably, the kidney cells are selected from 293 cells, such as 293T cells; and the ovarian cells are selected from CHO cells.

6. An exosome, characterized in that The exosomes comprise the composition according to claim 1 or 2 or are obtained by secretion from the transformant according to claim 5.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the composition according to claim 1 or 2, the recombinant expression vector or recombinant expression vector composition according to claim 3 or 4, the transformant according to claim 5, or the exosome according to claim 6; Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

8. A method for preparing the exosomes according to claim 6, characterized in that: The method comprises culturing the transformant according to claim 5, and obtaining exosomes from the culture.

9. A method for increasing the enrichment of Escherichia coli cytosine deaminase CD in exosomes, characterized in that: The method comprises combining a nucleotide encoding Escherichia coli cytosine deaminase CD or CD protein with a transmembrane protein CD63; Preferably, the recombinant expression vector or recombinant expression vector composition according to claim 3 or 4, or the transformant according to claim 5 is used; More preferably, the method comprises the following steps: A recombinant expression vector or a recombinant expression vector composition is prepared, the recombinant expression vector or the recombinant expression vector composition is transferred into mammalian cell culture, and exosomes are collected after culture; the recombinant expression vector or the recombinant expression vector composition is the recombinant expression vector or the recombinant expression vector composition defined in claim 3 or 4.

10. A method for inhibiting tumor cells, characterized in that: The method comprises contacting tumor cells with the composition according to claim 1 or 2, the exosomes according to claim 6, or the pharmaceutical composition according to claim 7; the method is for non-diagnostic treatment purposes.

11. Use of the composition according to claim 1 or 2, the recombinant expression vector or recombinant expression vector composition according to claim 3 or 4, the transformant according to claim 5, the exosomes according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a product for a cell proliferative disorder; Preferably, the cell proliferative disorder is selected from at least one of the following: non-small cell lung cancer, lung cancer, colon cancer, gastric cancer and pancreatic cancer.

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