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Specific sgRNA, expression vector and application of combined immune gene inhibition of HBV replication

A technology of gene suppression and expression vector, applied in the fields of genetic engineering and biomedicine, can solve the problems of high cost, expensive clinical anti-hepatitis B drugs, low efficiency, etc. Effect

Active Publication Date: 2020-02-07
李旭
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are following problems in the technical scheme for the treatment of hepatitis B now: (1) the drug effect is only to play a temporary blocking effect; (2) there are a lot of therapeutic drugs, how to utilize multiple treatment methods to jointly treat hepatitis B and there is no countermeasure; (3) efficiency Low, only a small amount of HBV cccDNA (covalently closed circular DNA) can be knocked out; (4) can only slightly reduce the expression of HBsAg; (5) due to the high cost of drug development, clinical anti-hepatitis B drugs are expensive, etc.

Method used

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  • Specific sgRNA, expression vector and application of combined immune gene inhibition of HBV replication
  • Specific sgRNA, expression vector and application of combined immune gene inhibition of HBV replication
  • Specific sgRNA, expression vector and application of combined immune gene inhibition of HBV replication

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preparation example Construction

[0097] 1. Preparation and culture of DC cells

[0098] (1) Kill the mice by neck breaking, immerse them in 75% alcohol for 5 minutes, separate and take out the two femurs and tibias of the mice under aseptic conditions, remove the muscles and fascia, sterilize in 75% alcohol for 30 seconds, and place them in normal saline spare;

[0099] (2) Use scissors to cut off the two ends of the tibia and femur, expose the bone marrow canal, use a 10mL syringe to absorb normal saline, insert it into both ends of the bone marrow, and flush out the bone marrow into a 50mL centrifuge tube;

[0100] (3) Centrifuge at 1500rpm for 10min, discard the supernatant, add 2mL erythrocyte lysate to precipitate, pipette for about 2min, then add about 40mL normal saline to precipitate and mix well;

[0101] (4) Centrifuge at 1000rpm for 10min, discard the supernatant, add 5mL of fresh 1640 culture medium, count the number of cells under the microscope, and use 1x10 6 mL cell density seeded into 6-wel...

specific Embodiment approach

[0133] Theoretically, after Cas9 cuts off the genome, the cell will repair the genome through non-homologous recombination end-joining. Because this repair mode is prone to mutations, processed genome sequences were tested experimentally as well as deep-sequencing experiments for this mutation. The specific implementation is as follows:

[0134] 1. The cell genome was extracted from the mouse liver, and the kit used was Tiangen Cell Blood Tissue Genome Extraction Kit, and the method was carried out according to the manufacturer's instructions.

[0135] 2. Obtain a fragment that includes the target site.

[0136] PCR reaction system

[0137]

[0138]

[0139] PCR reaction cycle:

[0140] 1) 5min at 95°C;

[0141] 2) 35 cycles

[0142] 95℃ 30s

[0143] 55℃ 30s

[0144] 68°C 30s

[0145] 3) 10min at 68°C

[0146] 3. Use the company's gel recovery kit to recover the target fragment. The specific method is carried out according to the test method provided by the com...

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Abstract

The invention provides a specific sgRNA combined with an immunogene to inhibit HBV replication, an expression vector thereof, and an application of the specific sgRNA and the expression vector. The sgRNA sequences of a human hepatitis B virogene suitable for CRISPR-Cas9 targeting editing and a PD-1 gene are designed, the plasmid vector of the sgRNA inhibiting HBV and PD-1 genes is constructed, and the plasmid vector and a nuclease gene expression vector are transferred to HBV transgenic mice, and can obviously inhibit HBV DNA replication. The gene expression vector prepared in the invention has the advantages of simple method steps, good sgRNA targeting property, and high CRISPR-Cas9 knockout efficiency. The sgRNAs specifically targeting the HBV and PD-1 genes can accurately splice the HBV and PD-1 genes, inhibit in vivo hepatitis B virus replication, and reduce the hepatitis B virus antigen expression.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biomedicine, and relates to the innovative design of CRISPR / Cas9 specifically modifying multiple gene targets of hepatitis B virus HBV and PD-1, and introduces combined immunotherapy strategies into the field of gene therapy to obtain enhanced targeted genes Good effect in the treatment of hepatitis B. Background technique [0002] In recent years, genome editing tools have been widely used in the field of biomedicine, and clustered regularly interspaced short palindromic repeats (CRISPR) technology has become a hot spot in genome editing. CRISPR is a sequence naturally present in bacterial DNA that binds to CRISPR-associated (Cas) nucleases that function as guide RNAs to protect the bacterial genome from attack by target-specific sequences detected in invasive phages. CRISPR / Cas9 technology was rated as one of the top 10 star technologies in 2013 by Nature and Science magazines, and ranked...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85A61K31/7088A61K48/00A61P1/16A61P31/20
CPCC07K14/70521C12N15/1131C12N15/1138C12N15/85C12N2310/10C12N2800/107C12N2800/80C12N2810/10
Inventor 甄帅李旭赵乐罗文娟
Owner 李旭
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