Survivin promoter mediated recombinant vector for expressing Gelonin phytotoxin genes and usage thereof

A technology of recombinant vectors and promoters, applied in the field of genetic engineering, can solve the problems of large differences in protein expression and low protein expression, and achieve the effects of inhibiting value growth, broad application prospects, and low toxicity

Inactive Publication Date: 2013-09-11
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the expression of survivin protein in paracancerous tissue

Method used

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  • Survivin promoter mediated recombinant vector for expressing Gelonin phytotoxin genes and usage thereof
  • Survivin promoter mediated recombinant vector for expressing Gelonin phytotoxin genes and usage thereof
  • Survivin promoter mediated recombinant vector for expressing Gelonin phytotoxin genes and usage thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Western blot detection of survivin protein expression

[0074] 1. Extraction of total protein

[0075] 1) Human normal liver cell line LO2, liver cancer cell line HepG2, SMMC-7721, and BEL-7402 cells in good growth state (purchased from the Cell Library of the National Academy of Sciences) were centrifuged and collected in a centrifuge tube after digestion with trypsin. At the same time, the suspended cells in the culture medium were collected together;

[0076] 2) Add RIPA lysate (purchased from Zhongshan Jinqiao Company, add about 400 microliters of RIPA to each bottle of cells) to resuspend the cells by pipetting repeatedly, add 10 microliters of phenylmethylsulfonyl fluoride (PMSF) (100 milliliters), shake well and place on ice;

[0077] 3) Lyse on ice for 30 minutes and mix from time to time;

[0078] 4) After lysis, centrifuge at 12,000 rpm for 5 minutes at 4°C;

[0079] 5) Transfer the centrifuged supernatant to a centrifuge tube for 0.5 minutes an...

Embodiment 2

[0097] Example 2 Vector construction

[0098] Since the cytomegalovirus promoter can mediate gene expression in both normal tissue and cancer tissue, we chose the cytomegalovirus promoter. The nucleic acid sequence is shown in SEQ ID No.8. As a control, PAAV was purchased from Cell Biolabs. -MCS expression vector named as pCMV-MCS. Afterwards, the survivin promoter sequence was synthesized by gene, amplified in vitro, and NotI and EcoRI restriction sites were introduced. The amplified survivin promoter and pCMV-MCS vector were subjected to double digestion, and the survivin promoter was connected to the vector, so that the cytomegalovirus promoter was replaced by the survivin promoter, and a pSur-MCS vector was constructed.

[0099] Green fluorescent protein is a reporter molecule that can emit green fluorescence when excited by light in the blue wavelength range, which can be observed under a fluorescent microscope. Due to the characteristic of autofluorescence, it has been...

Embodiment 3

[0122] Example 3 Detection of Survivin promoter activity

[0123] 1. Survivin promoter-mediated green fluorescent egg protein expression activity detection

[0124] Culture LO2 and HepG2 cells, spread six-well plate, about 2.5×10 5 per plate and cultured overnight at 37°C. Wait for the cell confluency to reach 50-60%. By mixing liposomes and plasmids, 2 micrograms of constructed pCMV-GFP and pSur-GFP recombinant plasmids were transiently transferred to each well of cells. Among them, the solid cationic liposome is prepared by our laboratory, and the preparation method of the solid liposome is to use chloroform and ethanol with a volume ratio of 3:1 to mix 1,2-dioleoyl-3- Trimethylaminopropane (DOTAP) and cholesterol (CHOL) were dissolved, and the lipid mixture was dried into a thin layer in a round-bottomed flask. Residual chloroform and ethanol were removed under high vacuum, and then the lipid was washed with 5% Glucose was hydrated, and the lipid was completely dissolve...

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Abstract

The invention belongs to the technical field of gene engineering, and in particular relates to a survivin promoter mediated recombinant vector for expressing Gelonin phytotoxin genes and usage of the recombinant vector, aiming to a method for searching safe and effective phytotoxin to treat liver cancer. The technical scheme of the invention is that a survivin promoter is operatively connected with a gene expression frame formed by the Gelonin phytotoxin genes. The invention also provides the survivin promoter mediated recombinant vector for expressing the Gelonin phytotoxin genes. A new method is provided for survivin promoter mediated phytotoxin target gene treatment, and a safe and effective cancer treatment new strategy is provided for a clinical test; and therefore, the recombinant vector has high application value and wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant vector for expressing a plant toxin Gelonin gene mediated by a Survivin promoter and an application thereof. Background technique [0002] Liver cancer is a malignant tumor that seriously threatens human health [1]. According to the latest statistics, there are about 600,000 new patients with liver cancer every year in the world [2], and the trend is increasing year by year. Liver cancer ranks third among death-related cancers [1]. The average survival time of patients diagnosed with advanced liver cancer is usually less than 3 months, and the five-year survival rate is only about 10% [3-5]. Therefore, finding effective treatment strategies and methods is a difficult and urgent problem for scientific researchers. Until now, researchers have not found a very ideal way to treat liver cancer. [0003] Previous studies have shown that toxins ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/85C12N5/10A61K48/00A61P35/00A61P1/16
Inventor 李炯魏于全杨莉周西坤王振张萍
Owner SICHUAN UNIV
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