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A kind of plasmid carrying nanoparticle and preparation method for preventing and treating influenza virus

A nanoparticle and plasmid technology, applied in biochemical equipment and methods, antiviral agents, gene therapy, etc., can solve the problems of target gene mutation, target gene inactivation, deletion, etc., and achieve the effect of great application value

Active Publication Date: 2022-04-05
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Non-homologous end joining can cause mutation of the target gene (insertion or deletion), which may inactivate the target gene

Method used

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  • A kind of plasmid carrying nanoparticle and preparation method for preventing and treating influenza virus
  • A kind of plasmid carrying nanoparticle and preparation method for preventing and treating influenza virus
  • A kind of plasmid carrying nanoparticle and preparation method for preventing and treating influenza virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] The preparation of embodiment 1 nanoparticle

[0147] 1. Experimental method

[0148] The synthetic route for the preparation of nanoparticles is shown in figure 1 shown.

[0149] 1. Synthesis of BLA-NCA monomer

[0150] (1) Synthesis of benzyl aspartate (BLA)

[0151] Prepare a single-necked bottle (500mL), add 100mL of anhydrous ether, then slowly add 10mL of H 2 SO 4 (98%), stirring vigorously while adding dropwise, after cooling to room temperature, add 100mL benzyl alcohol, after fully stirring, use the rotary evaporator to rotary evaporate ether. Add 13.3 g of aspartic acid (L-aspartic acid) in three times then to the reaction flask. Stir uniformly at room temperature for 24 hours, then add 200 mL of 95% ethanol, and add 50 mL of pyridine dropwise with a dropping funnel, stirring vigorously while dropping. Then freeze overnight in the refrigerator, pump the filtered solid, stir at 80°C, dissolve with deionized water, heat filter, and refrigerate the filtrat...

Embodiment 2

[0167] Example 2 Cellular uptake of nanoparticles

[0168] 1. Experimental method

[0169] 1. Mark the SP-dCas9-VPR plasmid

[0170] The plasmid SP-dCas9-VPR plasmid expressing dCas9 protein was incubated with YOYO-1 dye (Thermofisher) for 5 minutes, and the incubation ratio was carried out according to the official instructions, and then the SP-dCas9-VPR plasmid labeled with green fluorescent marker was obtained for subsequent cell uptake Experimental observation.

[0171] 2. Transfection of nanoparticles loaded with SP-dCas9-VPR plasmid

[0172] The SP-dCas9-VPR plasmid labeled with YOYO-1 and the Ba-PAsp(Pep-co-DIP)-Ac nanoparticles prepared in Example 1 were used to prepare the plasmid transfer carrier. The specific preparation process is as follows:

[0173] The polymer Ba-PAsp(Pep-co-DIP)-Ac was dissolved in a buffer solution of acetic acid and sodium acetate (PH=5.5, 0.1M) to 5 mg / mL, and the polymer solution and the plasmid solution were mixed at a mass ratio of 40 / ...

Embodiment 3

[0179] Example 3 Construction of up-regulated gRNA plasmid expressing IFNL gene

[0180] 1. Experimental method

[0181] 1. Design of gRNA

[0182] Query the target IFNL gene on the NCBI official website to obtain the full sequence. The first base of the obtained full sequence is the TSS site, the upstream query is -600bp, the downstream query is +100bp, and the obtained 700bp sequence is the gRNA target sequence. Design gRNA sequences, and design at least 5 gRNAs for each gene.

[0183] The target gene is the IFNⅢ gene, 4 members IFNL1 (IL-29), IFNL2 (IL-28a), IFNL3 (IL-28b), IFNL4 in humans, and 2 members IFNL2 (IL-28a) in mice , IFNL3 (IL-28b).

[0184] The gRNA is composed of an RNA sequence fragment that specifically recognizes the target region and a fixed backbone RNA sequence. The backbone sequence is: 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUUCAACUUGAAAAAGUGGCACCGAGUCGGUG-3'(SEQ ID NO:5). The gRNA sequences described later are all related to the target Re...

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Abstract

The invention discloses a plasmid carrying nanoparticle for preventing and treating influenza virus and a preparation method thereof. The plasmid-carrying nanoparticles include transport vectors and plasmids, and the plasmids include a combination of plasmids and SP-dCas9-VPR plasmids that are used to suppress H1N1 viruses in the CRISPRa system, and the transport vectors are polymeric plasmid transport vectors or lipids Plasmid transfer vector. The prepared CRISPRa plasmid system of the present invention up-regulates the expression of the IFNⅢ gene by the plasmid-carrying nanoparticles, which can be used to prevent influenza virus infection in advance and treat the initial stage of influenza virus infection. This kind of prevention or treatment for influenza virus is suitable for influenza virus subtypes. It is universal, has great application value, and is worthy of vigorous promotion.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to a plasmid-carrying nanoparticle for preventing and treating influenza virus and a preparation method thereof. Background technique [0002] Influenza virus, short for influenza virus, is an RNA virus. It is divided into three types: A (A), B (B), and C (C). The bovine influenza virus discovered in recent years will be classified as D (D). Influenza viruses are spread primarily through airborne droplets, contact between a susceptible person and an infected person, or contact with contaminated items. Human influenza is mainly caused by influenza A virus and influenza B virus. Influenza A virus is the most likely to mutate and is highly pathogenic to humans. The main symptoms after infection are high fever, cough, runny nose, myalgia, etc. Most of them are accompanied by severe pneumonia. Organ failure leads to death, and the case fatality rate is very high. Among them, the wel...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85A61K48/00A61P31/16
CPCC12N15/1131C12N15/85C12N15/111A61K48/005A61K48/0041A61P31/16C12N2310/20C12N2320/32C12N2800/107
Inventor 程度刘爽邓少辉
Owner SUN YAT SEN UNIV
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