Application of Stat3, HBx and Stat3-HBx target CRISPR/Cas carriers to preparation of HBV-related liver cancer drugs

A carrier, targeting technology, applied in the field of genetic engineering, can solve problems such as

Active Publication Date: 2020-01-07
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no high-efficiency vector for the Stat3 target, and a gene knockout product that can simultaneously knock out the two targets of Stat3-HBx

Method used

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  • Application of Stat3, HBx and Stat3-HBx target CRISPR/Cas carriers to preparation of HBV-related liver cancer drugs
  • Application of Stat3, HBx and Stat3-HBx target CRISPR/Cas carriers to preparation of HBV-related liver cancer drugs
  • Application of Stat3, HBx and Stat3-HBx target CRISPR/Cas carriers to preparation of HBV-related liver cancer drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Example 1: Construction of single-function silencing vectors targeting Stat3 and HBx respectively

[0095] Select the PX333 vector, digest it with BsaⅠ, and recover the linear fragment through agarose gel. According to the design strategy of CRISPR / Cas9 sgRNA, design 3 sgRNAs for the second and eighth exons of Stat3; for the gene sequence of HBx Design 2 sgRNAs (Table 1). The sgRNA fragments were annealed, ligated with the recovered fragments of the PX333 vector by T4 ligase, transformed into competent cells, and single clones were selected for sequencing. Sequencing results showed that 3 kinds of monofunctional plasmids targeting Stat3 and 2 kinds of targeting HBx were successfully constructed.

[0096] Table 1: sgRNA information

[0097]

[0098]

Embodiment 2

[0099] Example 2: Screening of a single-function silencing vector targeting Stat3

[0100] (1) Both Stat3-sgRNA1 and Stat3-sgRNA2 can reduce the expression of Stat3 protein

[0101] Experimental method: HepG2.2.15 cells use liposome Lipo6000 as a transfection reagent, set ctrl control, lipo6000 control (transfection reagent only), PX333 (vector control), and three plasmid vectors constructed to silence stat3, transfect After 48 hours of staining, the protein was extracted and detected by Western Blot.

[0102] Result: as attached figure 2 As shown, the results show that targeting both Stat3-sgRNA1 and sgRNA2 sequences can reduce the expression of Stat3 protein.

[0103] (2) Stat3-sgRNA1 and Stat3-sgRNA2 inhibit the proliferation of HepG2.2.15 cells

[0104] Methods: MTT and living cell monitor were used to study the proliferation of HepG2.2.15 cells after transfection of Stat3 plasmid.

[0105] Results: MTT results showed that targeting the Stat3-sgRNA1 sequence could inhib...

Embodiment 3

[0131] Example 3: Screening of HBx-targeting monofunctional silencing vectors

[0132] HBx-sgRNA2 inhibits the expression of HBx protein

[0133] Method: HepG2.2.15 cells use liposome Lipo6000 as transfection reagent, set ctrl control, lipo6000 control (transfection reagent only), PX333 (vector control), and two plasmid vectors constructed to silence HBx, and transfect After 48 hours, the protein was extracted, and the expression of HBx, p-Stat3, and Stat3 proteins were detected by Western Blot. The results are shown in the attached Figure 8 shown.

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Abstract

The invention discloses a Stat3 target CRISPR / Cas carrier, a Stat3-HBx dual-target CRISPR / Cas carrier, and two repeated-target CRISPR / Cas carriers targeting HBx, and particularly relates to an Stat3 target single-function plasmid, an Stat3-HBx dual-target CRISPR / Cas carrier, two CRISPR / Cas carriers repeatedly targeting the HBx, and a construction method of the carriers. Through tests, the inventorproves that the constructed carriers have a specific target treatment effect on HBV-related liver cancer cells, and the silent gene carrier, the carrier construction method, double-silence strategiesof the carriers for Stat3 and the HBx and application of the carriers to preparation of HBV chronic infection and anti-liver cancer related drugs are within the scope of right protection correspondingly.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to the application of Stat3 and HBx target gene CRISPR / Cas silencing vectors in inhibiting HBV chronic infection and treating HBV-related liver cancer. Background technique [0002] Hepatocellular carcinoma is one of the five most common cancers in the world and ranks the third cause of cancer death. In clinical treatment, the five-year survival rate of liver cancer patients is only about 10%. According to the latest 2015 Chinese cancer statistics, liver cancer is the most common cancer diagnosed before the age of 60, and accounts for the first cancer death rate in men, followed by lung cancer and stomach cancer. HBV infection has been considered as an important risk factor for HCC. In many tumors such as liver cancer, there is abnormal expression and persistent activation of signal transducers and activators of transcription 3 (STAT3), and its target genes i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90A61K31/713A61P35/00A61P31/20
CPCA61K31/713A61P31/20A61P35/00C07K14/005C07K14/4702C12N15/113C12N15/85C12N15/907C12N2310/10C12N2730/10122C12N2310/20
Inventor 张建郭全娟韩秋菊
Owner SHANDONG UNIV
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