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Recombinant adeno-associated virus vector of tumour-targeted double genes

A virus carrier and gene technology, applied in gene therapy, antineoplastic drugs, recombinant DNA technology, etc., can solve problems such as undiscovered disease reports, and achieve the effect of improving therapeutic effect and obvious anticancer effect

Active Publication Date: 2013-12-18
DONGGUAN ZHENGXING BEITE MEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no disease related to AAV has been reported, so AAV is recognized as the safest viral vector

Method used

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  • Recombinant adeno-associated virus vector of tumour-targeted double genes
  • Recombinant adeno-associated virus vector of tumour-targeted double genes
  • Recombinant adeno-associated virus vector of tumour-targeted double genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Construction of recombinant adeno-associated virus vector pAAV-Rev-Caspase3-IRES-Endostatin plasmid

[0035] 1) Primers for RT-PCR cloning Rev-Caspase3 gene:

[0036] Rev-Caspase3 is divided into large subunit LS (p17) and small subunit SS (p12). According to the sequence, primers were designed with P5 software as follows:

[0037] LS(p17):

[0038]

[0039] Wherein BamH I is added upstream, and Xho I restriction site sequence is added downstream (as shown in the box).

[0040] SS(p12):

[0041]

[0042] Among them, EcoR I is added upstream, and BamH I restriction site sequence is added downstream (as shown in the box).

[0043] The total RNA of A549 cells (cell source: purchased from the Cell Bank of Experimental Animal Center of Sun Yat-sen University, Guangzhou) was extracted with Trizol, and cDNA was synthesized by reverse transcription using this as a template, and the whole caspase-3 gene was amplified by PCR with P1 and P4 primers, and then P...

Embodiment 2

[0062] Example 2. Preparation of recombinant adeno-associated virus vector rAAV-Rev-Caspase3-IRES-Endostatin

[0063] HEK293 cells (cell source: ATCC, USA) were used as packaging cells, subcultured with DMEM medium and 10% newborn calf serum, and the cells grew to 80%-85% confluence, and the auxiliary plasmids pAAV2-RC, pHelper (purchased from Stratagene, USA) and pAAV-Rev-Caspase3-IRES-Endostatin three plasmids constructed in Example 1 were co-transfected into HEK293 cells, and the transfection solution was configured according to the transfection of 10 dishes: 10 μg / dish of various plasmids ( 10cm), 1.5mol / L CaCl 2 2ml, add sterilized double distilled water to 15ml and mix well, then add 15ml of buffer A (50mM Hepes, 1.5mM Na 2 HPO 4 , 280mM NaCl, pH 7.05), mix well and let it stand for 5 minutes, then start to transfect 293 cells, 3ml / dish, continue to place in a carbon dioxide incubator with 5% CO 2 , 37°C and saturated humidity for 65-70 hours, collect the cells and s...

Embodiment 3

[0071] Example 3. Verification of anti-tumor effect of rAAV-Rev-Caspase3-IRES-Endostatin recombinant virus

[0072] 1) Cell test results:

[0073] Use the virus that embodiment 2 obtains according to 5 * 10 4 v.g / cell was transfected with HepG-2 cells (purchased from the Cell Bank of the Experimental Animal Center of Sun Yat-Sen University, Guangzhou). After 24 hours, the number of cells observed under the ordinary light microscope was significantly reduced compared with the control (untransfected virus), and after 48 and 72 hours Observed under the Ruiji staining microscope, compared with the control, the number of cells after transfection with the virus was significantly reduced, and the cytoplasm became larger, with nuclear pyknosis (see Figure 3).

[0074] The results showed that the virus had obvious inhibitory effect on the growth of liver cancer HepG-2 cells.

[0075] 2) Animal test results:

[0076] The recombinant adeno-associated virus rAAV-Rev-Caspase3-IRES-Endosta...

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Abstract

The invention relates to a recombinant adeno-associated virus vector of tumour-targeted double genes, in particular to a recombinant adeno-associated virus vector containing ribosomal reverse connected Caspase 3 and endostatin simultaneously, and a preparation method and anti-tumour application of the vector.

Description

technical field [0001] The invention relates to the fields of virology, immunology, gene proteinology and biomedicine, in particular to a double gene-containing recombinant adeno-associated virus vector, its preparation method and its application. Background technique [0002] Malignant tumors are currently one of the major diseases that seriously affect human health and threaten human life. At present, radiotherapy and chemotherapy are mainly used clinically. However, some malignant tumors are not sensitive to radiotherapy and are prone to drug resistance to chemotherapy. Therefore, it is an urgent desire for clinicians and patients to find new candidate drugs and therapeutic methods for effective treatment of tumors. The study of tumor molecular biology shows that tumor cell apoptosis is a key link in tumor development. Therefore, the strategy of inducing tumor cell apoptosis to achieve the purpose of tumor suppression has become a hot spot in tumor gene therapy in recen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/86C12N5/10A61K48/00A61P35/00
Inventor 谭孟群赵霏唐升斌肖艳桃
Owner DONGGUAN ZHENGXING BEITE MEDICINE TECH CO LTD
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