Manipulation system for regulating and controlling specific expression of suicide gene in liver tumor cells

A suicide gene and liver tumor technology, applied in gene therapy, antineoplastic drugs, genetic engineering, etc., can solve the problem of poor targeting of liver cancer cells, low selectivity of therapeutic gene expression, and adenovirus self-replicating characteristics targeting cells Uncontrollability and other issues

Inactive Publication Date: 2018-09-07
邹卫龙
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the self-replicating properties of adenovirus and the uncontrollability of targeting cells limit the effective application of this system in clinic
[0004] Not only that, most suicide gene therapy for liver cancer is in the stage of animal and clinical trials, and the main problems restricting its promotion are safety and effectiveness, including low selectivity of therapeutic gene expression, weak targeting of liver cancer cells, suicide gene Or the carrier itself will produce toxic side effects on normal liver cells, etc.

Method used

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  • Manipulation system for regulating and controlling specific expression of suicide gene in liver tumor cells
  • Manipulation system for regulating and controlling specific expression of suicide gene in liver tumor cells
  • Manipulation system for regulating and controlling specific expression of suicide gene in liver tumor cells

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Embodiment 1

[0039] This embodiment is used to illustrate the construction of the system of the present invention.

[0040] 1. Construction of rAAV-Survivin-LoxP-Stop-LoxP-TK vector

[0041] The TK gene sequence (Gene ID: 7083) was synthesized according to the information published by NCBI, and the SURV promoter (Survivin) was synthesized according to the sequence shown in SEQ ID NO.1.

[0042] The SURV promoter and TK gene were constructed into plasmid CN1005 (purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.) using restriction enzymes XbaI and KpnI, and restriction enzymes BamHI and HindIII, respectively, to obtain rAAV- Survivin-LoxP-Stop-LoxP-TK vector, map as figure 1 As shown in A.

[0043] The enzyme cleavage and ligation methods can adopt conventional means and conditions in the art.

[0044] 2. Construction of rAAV-AFP-Cre vector

[0045] AFP promoter (Gene ID: 174) and Cre gene sequence (Gene ID: 2777477) were synthesized according to the information published by NCBI, a...

Embodiment 2

[0047] This example is used to illustrate that based on the principle of the Cre-LoxP recombination system, the Cre recombinase driven by the AFP promoter of rAAV exerts site-specific and controllable knockout of the Survivin promoter to drive the STOP of the LoxP open reading frame inserted into the TK suicide gene Intron, can drive the expression of the downstream suicide gene TK.

[0048] 1. Co-infect HpG2 cells and Huh7 cells with rAAV-AFP-Cre (also called H5312) and rAAV-SURV-LoxP-Stop-LoxP-TK (also called H5315) at different MOIs, and detect the expression of TK gene. The dosage of virus is as follows: 1#MOI 1*10 5 , that is, H5312 and H5315 viruses each use 0.5*10 5 ;2#MOI 2*10 5 , that is, H5312 and H5315 viruses each use 1*10 5 ;3#MOI 1*10 6 , that is, H5312 and H5315 viruses each use 5*10 5 . The result is as figure 2 As shown, the rAAV-AFP-Cre and rAAV-SURV-LoxP-Stop-LoxP-TK operating systems can drive the expression of TK, and the expression level is depend...

Embodiment 3

[0052] Infect Huh7 cells with rAAV-AFP-Cre / rAAV-SURV-LoxP-Stop-LoxP-TK and control AOV032 (purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.) to detect the killing of TK / GCV and the amount of virus For MOI 5*10 6 , rAAV-AFP-Cre and rAAV-SURV-LoxP-Stop-LoxP-TK viruses each use 2.5*10 6 . The result is as Figure 4 As shown, in Huh7 cells, after virus infection, under the action of GCV, the cell survival rate gradually decreased with the increase of GCV drug concentration, showing a dose-effect-dependent relationship. In the GCV2ug / ml and 10ug / ml drug concentration groups , the p value is less than 0.05, which is statistically significant.

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Abstract

The invention belongs to a biotechnology and in particular discloses a manipulation system for regulating and controlling specific expression of a suicide gene in liver tumor cells. The manipulation system comprises: (1) a Cre recombinase gene which is driven by an AFP promoter to express; (2) the suicide gene which is driven by an SURV promoter to express, wherein one segment of LoxP-Stop-LoxP sequence is inserted between the SURV promoter and the suicide gene, Stop is a transcription termination sequence and LoxP is a specific recognition sequence of Cre recombinase. According to the manipulation system disclosed by the invention, the AFP promoter is used for driving the expression of the Cre recombinase in liver cancer HepG2 cells; the Cre recombinase is used for cutting off the transcription termination sequence Stop between the two LoxP so that the tumor specific SURV promoter can be used for directly driving expression of TK/GCV; liver tumor cell apoptosis is specifically induced, but cells with any other types in bodies are not injured.

Description

technical field [0001] The invention belongs to biotechnology, and in particular relates to an operating system for regulating the high-efficiency and specific expression of suicide genes in liver tumor cells. Background technique [0002] The morbidity and disease-related mortality of hepatocellular carcinoma (Hepatocellular carcinoma, HCC) ranks 6th and 3rd in the global cancer survey respectively. More than 55% of the 700,000 new cases of liver cancer in the world every year are in my country. Treated patients usually die within 3 to 6 months after diagnosis. Liver resection and liver transplantation are currently the main curative treatments for HCC. However, 40% of patients with negative margins of liver resection will relapse within one year after surgery; even following the most stringent selection criteria (ie Milan criteria: single Tumor diameter ≤ 5 cm, or multiple tumors ≤ 3 and the largest diameter ≤ 3 cm, without large vessel invasion or extrahepatic metastasis)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/864A61K48/00A61K38/45A61P35/00
CPCA61K38/45A61K48/0058A61P35/00C12N15/86C12N2750/14143C12N2800/107C12N2800/30C12Y207/01021
Inventor 邹卫龙童畅
Owner 邹卫龙
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