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Recombinant plasmid and virus of tissue specificity and application thereof.

A technology of recombining plasmids and viruses, applied in the fields of application, virus/bacteriophage, recombinant DNA technology, etc., can solve the problems of toxicity, inability to distinguish tumor cells and normal cells, etc., to avoid influence, good clinical application prospects, and avoid normal cells. The effect of cellular influence

Inactive Publication Date: 2012-07-04
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since cantharidin can not distinguish tumor cells from normal cells when it kills cells by inhibiting PP2Ac, cantharidin also has a toxic effect on normal cells and tissues

Method used

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  • Recombinant plasmid and virus of tissue specificity and application thereof.
  • Recombinant plasmid and virus of tissue specificity and application thereof.
  • Recombinant plasmid and virus of tissue specificity and application thereof.

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Cloning PP2Ac and mutating it into DN-PP2Ac by site-directed mutagenesis

[0046] The inventor has constructed the PP2Ac expression plasmid pcDNA3.1-PP2Acα in previous studies (see Li W, et al.. Cancer Lett 2011; 304: 117-27), and the plasmid map is as follows figure 1 shown. The PP2Ac gene sequence is shown in SEQ ID No.4 or SEQ ID No.5.

[0047] The DN-PP2Ac expression plasmid was obtained from pcDNA3.1-PP2Acα by site-directed mutagenesis. The mutated plasmid was named pcDNA3.1-DN-PP2Acα. It was confirmed by sequencing that the 199th leucine (CTG) of PP2Acα had been mutated to proline (CCG). The amino acid sequence of PP2Acα is shown in SEQ ID No.6, and the amino acid sequence of the negative dominant mutant DN-PP2Acα expressed after mutation is shown in SEQ ID No.1.

Embodiment 2

[0048] Example 2: Functional identification of DN-PP2Ac

[0049] A protein phosphatase activity detection kit (Nonradioactive serine / threonine-phosphatase assay kit, purchased from Promega) was used to detect the inhibitory effect of DN-PP2Ac expression on PP2A activity. The normal human liver cell line L-02, and the liver cancer cell lines SK-Hep-1 and HepG2 were transfected with pcDNA3.1-DN-PP2Acα, and the results were as follows: figure 2 As shown, after transfection of pcDNA3.1-DN-PP2Acα into human liver cell line L-02, and liver cancer cell lines SK-Hep-1 and HepG2, its PP2A activity was significantly inhibited, and the difference was statistically significant compared with the control group .

[0050] The relative PP2A activity (relative PP2A activity) was calculated according to the following formula. Relative activity of PP2A=(PP2A activity of treatment group / PP2A activity of control group)×100%.

[0051] MTT assay was used to detect the inhibitory effect of DN-PP2...

Embodiment 3

[0054] Embodiment 3: Construction of AFpg promoter

[0055] AFP enhancer has tissue specificity but not strong transcriptional activity; pgk promoter has strong transcriptional activity but not tissue specificity. The combination of the two can have tissue specificity and strong transcriptional activity at the same time. The nucleotide sequence of the AFP enhancer is shown in SEQ ID No.2, and the nucleotide sequence of the pgk promoter is shown in SEQ ID No.3.

[0056] Using genomic DNA of liver cancer cell HepG2 as a template, the AFP enhancer and pgk promoter were cloned by PCR, and the two were constructed into the luciferase reporter gene vector pGL3-Basic (purchased from Promega Company), and the resulting plasmid was named pGL3-Basic- AFpg. Plasmid map such as Figure 4 shown.

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Abstract

The invention relates to the biotechnology field, and discloses a recombinant plasmid, a recombinant virus plasmid, a recombinant virus of tissue specificity directing against hepatoma cells with positive AFP (Alpha Fetal Protein) and application in preparing drugs for treatment of hepatoma with positive AFP. In the invention, DN-PP2Ac expression is regulated by AFpg promoter of tissue specificity of hepatoma, so that DN-PP2Ac specificity is expressed in hepatoma cells with positive AFP, thereby preventing the effect on normal cells and having a good prospect of clinical application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a tissue-specific recombinant plasmid, virus and application thereof for AFP-positive liver cancer cells. Background technique [0002] Liver cancer is a malignant tumor that occurs in the liver. It is one of the most common malignant tumors in clinical practice, ranking fifth among malignant tumors. Liver cancer is a high incidence rate in my country, generally more men than women. At present, the incidence of liver cancer in my country accounts for more than half of the world's total, accounting for 55% of global liver cancer patients. It has become a major killer that seriously threatens the health and life of our people, and its danger cannot be underestimated. [0003] Although cantharidin extracted from traditional Chinese medicine has a significant effect in treating liver cancer, it has obvious toxic and side effects on normal tissues due to its non-tissue specificity. The ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/861C12N15/867C12N7/01A61K48/00A61P35/00
Inventor 李伟龚斐然陶敏
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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