Survivin promoter-controlled suicide gene HSVtk eukaryotic expression vector

A eukaryotic expression vector, pbi-cmv2-acgfp1-psurvivin-tk technology, applied in the field of genetic engineering, can solve the problems of unsatisfactory treatment effect of advanced tumors, toxic side effects, and large number of patients, achieving good selectivity and low toxicity, Highly specific and selective effects

Active Publication Date: 2016-09-21
SHANXI MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since most patients are diagnosed at an advanced stage, traditional treatment methods are not ideal for the treatment of advanced tumors with recurrence and metastasis, and will cause relatively large toxic and side effects to patients.

Method used

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  • Survivin promoter-controlled suicide gene HSVtk eukaryotic expression vector
  • Survivin promoter-controlled suicide gene HSVtk eukaryotic expression vector
  • Survivin promoter-controlled suicide gene HSVtk eukaryotic expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: pBI-CMV 2 -AcGFP 1 - Construction of pSurvivin-TK eukaryotic expression vector.

[0025] 1. Extract HepG 2 Genomic DNA of liver cancer cells.

[0026] Cultivate HepG 2 Cells, when the cell growth density reaches 80%, collect the cells, wash twice with PBS buffer, extract genomic DNA with a DNA extraction and amplification kit, add 200 μl Buffer SA and 10 μl Proteinase K to mix, incubate at 56°C for 10 minutes, and then incubate at 95°C Incubate for 5 minutes, centrifuge at 13000rpm for 5 minutes, transfer the supernatant to a 1.5ml EP tube to obtain genomic DNA.

[0027] 2. PCR amplification of the Survivin promoter sequence.

[0028] Reaction system: 2×PCR Master Mix, 10 μl; 1 μl of upstream primer and downstream primer; 2 μl of genomic DNA; 6 μl of RNase-Free Water. Reaction conditions: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 40 s, annealing at 65°C for 30 s, extension at 72°C for 40 s, a total of 30 cycles, and final extension at...

Embodiment 2

[0044] Example 2: Eukaryotic expression vectors in KYSE150 esophageal cancer cells and HepG 2 Transfection efficiency in hepatoma cells.

[0045] Via Lipofectamine 2000 TM Mediated, pBI-CMV 2 -AcGFP 1 -pSurvivin-TK eukaryotic expression vector was transfected into KYSE150 esophageal cancer cells and HepG 2 For liver cancer cells, transfect for 48 hours, digest and collect the cells, resuspend the cells in PBS, detect the cells expressing EGFP by flow cytometry, and calculate the percentage of cells expressing EGFP. The transfection efficiency of the eukaryotic expression vector to the two cells was determined by the percentage of cells expressing EGFP in each group (transfection rate = number of green fluorescent cells / total number of cells × 100%).

[0046] image 3 Flow cytometric detection of eukaryotic expression vectors in KYSE150 esophageal carcinoma cells and HepG 2 Transfection efficiency results in hepatoma cells. In the figure: A is the detection result of gre...

Embodiment 3

[0049] Embodiment 3: Western blot method detection HSVtk Protein expression of suicide genes.

[0050] KYSE150 esophageal cancer cells and HepG 2 Liver cancer cells were seeded in 6-well plates and cultured. When the cells grow to 70-80% confluence, refer to Lipofectamine 2000 TM instructions for transfection. The old culture medium of the cells in the six-well plate was replaced with a serum-free medium, and the liposome and plasmid complexes were evenly added to the corresponding wells, and after 6 hours, the medium was changed to a medium containing 10% serum to continue culturing. After 48 hours of transfection, discard the cell culture medium, wash twice with PBS, add 150 μl of RIPA lysate and protease inhibitor PMSF (RIPA:PMSF=100:1) to each well, fully lyse and transfer to a 1.5ml centrifuge tube, 12000rpm Centrifuge for 10 minutes, take the supernatant, and transfer it to a new centrifuge tube.

[0051] Prepare the working solution as A:B=50:1, mix well; dilute th...

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Abstract

The invention discloses a Survivin promoter-controlled suicide gene HSVtk eukaryotic expression vector. The eukaryotic expression vector disclosed by the invention, in combination with an exogenous drug, namely ganciclovir (GCV), has a specific killing effect on KYSE150 esophageal carcinoma cells, and the effect is significantly stronger than that on HepG2 hepatoma carcinoma cells.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and relates to a suicide gene controlled by a Survivin promoter HSVtk The eukaryotic expression vector has a specific killing effect on KYSE150 esophageal cancer cells. Background technique [0002] Esophageal cancer is one of the common malignant tumors of the digestive tract, and the occurrence of this tumor is related to differences in regions, races, and pathological types. About 200,000 people die from esophageal cancer every year in the world. my country is a country with a high incidence of esophageal cancer, second only to gastric cancer in tumor mortality. The age of onset is mostly between 40 and 60 years old, and men are more than women. The current treatment strategies for esophageal cancer include surgery, radiotherapy, and chemotherapy, and surgery is the most important treatment. However, since most patients are diagnosed at an advanced stage, traditional treatment met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/54A61K38/45A61P35/00
CPCA61K38/45C12N9/1211C12N15/85C12N2800/107C12N2830/008C12Y207/01021
Inventor 于保锋李勇芳孟凡秀张琪齐永利李卓奇赵娜张英敏袁洋洋曾思衡姚志坚杨巧艳温晓薇温丽敏白欣艳张紫燕解军徐钧郭睿张悦红王惠珍弓韬王海龙杨丽红刁海鹏陈显久秦琴董秀山胡晓年
Owner SHANXI MEDICAL UNIV
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