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Tumor suppressing polypeptide TSF and gene therapy vector composition

A technology of composition and polypeptide substances, applied in gene therapy, drug combination, antineoplastic drugs, etc., can solve the problems of preparation process limitations, pharmacokinetics, etc., and achieve the effect of eliminating interference

Inactive Publication Date: 2003-03-12
TAIDA RES CENT OF LIFE SCI & TECH INST OF HEMATOLOGY CHINES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the limitations of the preparation process and the characteristics of pharmacokinetics, it is still difficult for the drugs in clinical trials to meet the above requirements.

Method used

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  • Tumor suppressing polypeptide TSF and gene therapy vector composition
  • Tumor suppressing polypeptide TSF and gene therapy vector composition
  • Tumor suppressing polypeptide TSF and gene therapy vector composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0006] Some examples are listed below to illustrate the present invention in detail, but not to limit the present invention. Example 1. Cloning of TSF cDNA and packaging of recombinant adenovirus Total RNA was extracted from normal human peripheral blood cells using TRIZOL kit (product of Life Technologies). TSF cDNA was amplified by RT-PCR, and the PCR reaction conditions were pre-denaturation at 94°C, 30”; annealing at 58°C, 45”; extension at 72°C, 2’, 30 cycles. The amplified product is 1.7Kb in length. The amplified product was detected by an automatic DNA sequencer, and the amplified product was completely consistent with the designed sequence. The PCR primers used were designed according to the full-length cDNA sequence of TSP1 (GENE BANK Accession Code NM003246), specifically: the upstream primer 5'-AAT CGA GGT ACC GTA CAC ACA GGA TCC CTG CTG-3'; the downstream primer 5'-GAT CCC AAG CTT TTA AAT TGG ACA GTC CTG CTT GTT-3'. The TSF cDNA coding region is 40-1683.

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Embodiment 2

[0008] The human leukemia cell line K562 was infected with TSF recombinant adenovirus, control virus and PBS, and the MOI of infection was 100. After 48 hours, the cells were collected to extract total RNA with TRIZOL kit (product of Life Technologies Company), and the One step RT- PCR kit was used to detect the expression of TSF at the transcriptional level in K562 cells ( figure 2 ). The PCR reaction conditions and primers used are as described in Example 1. At the same time, the culture supernatant of K562 cells 48 hours after virus infection was collected to detect the expression of TSF protein level by Western blotting. Specifically, after the culture supernatant was concentrated, the total protein was separated by 9% SDS-polyacrylamide gel electrophoresis, and the latter was electrotransferred to a PVDF membrane. The antibody was incubated with PVDF membrane, and DAB (product of Sigma company) was developed to detect the target protein ( image 3 ). The results of R...

Embodiment 3

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Abstract

A novel recombinant adenovirus vector mediated antineoplastic composition is prepared through cloning the cDNA sequence from the human peripheral blood cell by specific primer and PT-PCR method for coding TSF polypeptide, construction in human embryonic kidney cell 293 by AdEasy system, and packaging and expressing the recombinant adenovirus vector of TSF. It can suppress the growth and transfer of cancer.

Description

Technical field: [0001] The invention relates to a new anti-tumor gene therapy composition mediated by a recombinant adenovirus vector, which has the effect of targeting anti-angiogenesis in tumor tissue and inhibiting the growth and metastasis of tumors including hematopoietic malignant tumors. Specifically, it relates to the preparation and application in vivo and in vitro of a recombinant adenoviral vector expressing a new anti-tumor polypeptide TSF. Background technique: [0002] Tumor is a malignant disease of abnormal differentiation and proliferation of human cells. Regardless of primary or metastatic tumors, their continuous growth must rely on the formation of new blood vessels. The new capillary network provides tumor cells with the oxygen and nutrients needed for rapid growth and carries away metabolic waste. The diameter of solid tumors lacking blood supply can usually only be maintained within 1-2mm. Similar to the situation of solid tumors, recent studies ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/16A61K48/00A61P35/00
Inventor 韩忠朝刘澎
Owner TAIDA RES CENT OF LIFE SCI & TECH INST OF HEMATOLOGY CHINES