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Preparation method and application of carbon nanomaterial-immunostimulatory sequence compound

A technology of immunostimulatory sequences and carbon nanomaterials, applied in the biological field, can solve the problems of low cell uptake rate of immune stimulatory sequences, achieve good medical application prospects, save costs, and reduce pain

Active Publication Date: 2013-07-24
SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Therefore, the technical problem to be solved by the present invention is to address the defects that the existing immune stimulatory sequence (CpG) itself is easy to degrade and has a low cell uptake rate, and requires repeated administration of high doses to stimulate the body to produce an effective immune response. Preparation method and application of carbon nanomaterial-immunostimulatory sequence complex

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  • Preparation method and application of carbon nanomaterial-immunostimulatory sequence compound
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  • Preparation method and application of carbon nanomaterial-immunostimulatory sequence compound

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Embodiment 1

[0036] Embodiment 1 synthetic immunostimulatory sequence

[0037] All immunostimulatory sequence nucleotides were synthesized by Invitrogen, in which ssCpG is a single-stranded CpG oligodeoxynucleotide, nonCpG is an oligonucleotide without a CpG motif, and Cy3-CpG is a red fluorescently labeled oligonucleotide Nucleotides, S-CpG is an oligonucleotide containing a phosphorothioate backbone, all oligonucleotides have a phosphodiester bond backbone, and the sequences of the oligonucleotides are shown in Table 1 and sequence respectively. Shown in the list as SEQ ID NO: 1 to SEQ ID NO: 4.

[0038] The immunostimulatory sequence involved in the embodiment of table 1

[0039]

Embodiment 2

[0040] Example 2 Preparation of Nanodiamond Immune Sequence Complex (NDs-CpG Complex)

[0041] Polylysine modified nanodiamonds (see L.C. L. Huang and H. C. Chang. Adsorption and Immobilization of Cytochrome c on Nanodiamonds. Langmuir2004, 20, 5879-5884 for details).

[0042] Mix 70mg of nano-diamonds (NDs) (Gansu Jinshi Nano Materials Co., Ltd., the nano-diamonds have a purity of >99% and a single particle diameter of about 2-10nm, forming a cluster structure of about 250nm in the solution) and 30mg of poly Add polylysine (PDL) (sigma, product number: P7280) to 10 mL of boric acid, adjust the pH to 8.5 with 1M NaOH, mix well by ultrasonic, shake on a shaker at 25oC overnight, centrifuge at 13000rpm for 20min, and wash the obtained precipitate with millipore pure water for 5 After several times, the free PDL was completely removed, and the PDL-modified NDs (NDs-PDL) were obtained by centrifugation.

[0043] NDs-PDL and single-chain CpG (ssCpG) were mixed in millipore pure wa...

Embodiment 3

[0044] Example 3 Observation of Cellular Uptake Behavior of NDs-CpG

[0045] The Cy3-CpG (Cy3 exhibits red fluorescence) oligonucleotide synthesized in Example 1 was adsorbed to the NDs-PDL prepared in Example 2, and the preparation method was the same as in Example 2 to prepare fluorescently labeled NDs-CpG.

[0046] Raw264.7 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and cultured in DMEM medium (Gibco) containing 10% fetal bovine serum. 37°C, 5% CO 2 , cultured in saturated humidity. Cells at 5 x 10 4 / well density inoculated on laser confocal culture dish, adhered to the wall overnight. Set up the following experimental groups: blank control, ssCpG (5 μg / mL), NDs-PDL (50 μg / mL), NDs-CpG (50 μg / mL), incubate at 37°C for 6 h, wash with PBS, image with Leica TCS sp5 confocal fluorescence microscope (excitation wavelength 561nm, emission wavelength 620nm), 20-25 cells were randomly selected in each group, and the average fluorescence...

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Abstract

The invention discloses a preparation method and application of a carbon nanomaterial-immunostimulatory sequence compound. The preparation method comprises the steps of (1) modifying a carbon nanomaterial through polylysine to obtain a polylysine-modified carbon nanomaterial; and (2) mixing the polylysine-modified carbon nanomaterial obtained from the step (1) with an immunostimulatory sequence in a water solution, oscillating for 0.5-3 hours at 20-37 DEG C, and centrifuging, collecting and depositing to obtain the carbon nanomaterial-immunostimulatory sequence compound. According to the carbon nanomaterial-immunostimulatory sequence compound, the carbon nanomaterial serves as a carrier for intracellular transport of the immunostimulatory sequence (CpG DNA), so as to remarkably improve the cellular uptake efficiency of the CpG DNA, protect the CpG DNA from being degraded by nuclease and enhance the immunocompetence of an organism for a long time, so the carbon nanomaterial-immunostimulatory sequence compound has a good medical application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a carbon nanomaterial-immune stimulation sequence complex. Background technique [0002] CpG motifs (CpG motifs) refer to the nucleotides of the core sequence containing unmethylated cytosine (C) and guanine (G). A DNA sequence containing a CpG motif is called CpG DNA, or CpG ODN (oligodeoxynucleotide, oligodeoxynucleotide), also known as an immunostimulatory sequence. This kind of ODN containing unmethylated CpG dinucleotides occurs more frequently in bacterial and viral genomes than in vertebrates, and 80% of cytosines in vertebrates are methylated. Significant differences allow vertebrates to recognize invading bacteria and viruses, and thus CpG motifs are the basis of mammalian immunostimulatory signals. The artificially synthesized ODN sequence containing CpG motif can imitate the stimulating effect of bacterial DNA, activate...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K47/34A61K47/04A61K39/39A61K31/7088A61P37/02A61P31/00A61P35/00A61P37/08
Inventor 诸颖张瑜崔之芬孔华庭孙艳红黄庆樊春海
Owner SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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