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Recombination tumor chalone-tumor putrescence factor secretion type eukaryon expression vector and its preparation method and use

A technology of tumor necrosis factor and eukaryotic expression vector, which can be used in the field of medicine and biology and can solve problems such as dose limitation

Inactive Publication Date: 2008-09-17
罗以勤 +2
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Problems solved by technology

However, normal vascular endothelial cells are also affected by TNF, which is likely the reason for dose limitation due to hypotension and TNF toxicity

Method used

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  • Recombination tumor chalone-tumor putrescence factor secretion type eukaryon expression vector and its preparation method and use
  • Recombination tumor chalone-tumor putrescence factor secretion type eukaryon expression vector and its preparation method and use
  • Recombination tumor chalone-tumor putrescence factor secretion type eukaryon expression vector and its preparation method and use

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Embodiment Construction

[0039] (1) Experimental materials

[0040] 1.1 Strains, cells and plasmids: Escherichia coli DH5α, PBV220-TNF plasmid vector, human embryonic kidney cell line 293 cells were all preserved in our laboratory, pGEM-T was purchased from Promega Company, plasmid pIRESneo3 was purchased from Clontech Company, human embryonic kidney cell line 293 cells were cultured in 1640 containing 10% newborn calf serum, CHO-K1 cells were cultured in DMEM containing 10% fetal bovine serum, protein electrophoresis (Phamarcia Hoefer Biotech.Ltd.).

[0041] 1.2 Main reagents Small amount of plasmid extraction kit, DNA gel recovery kit, and total cell RNA extraction kit were purchased from Huashun Bioengineering Company; RT-PCR kit, PCR kit, various restriction enzymes, DNA mark and Ligase was purchased from TaKaRa Company. Trizol, Superscript II reverse transcriptase, G418 and lipofectamine TM 2000 was purchased from Invitrogen. Primers were synthesized by Shanghai Sangon Company. Fetal bovine s...

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Abstract

The invention relates to a secretion type eucaryon expression vector for recombining tumor chalone-tumor necrosis factor and a preparation method and the application thereof, and discloses the secretion type eucaryon expression vector of the tumor chalone (tumstatin) and human tumor necrosis factor (TNF-Alpha) and the method of preparing fused protein. Tumstatin-TNF can respectively interact with an AlphavBeta3 receptor on the surface of a neovascularization endothelial cell and a tumor cell death receptor in a highly specific way. A fractional step cloning method is adopted to construct secretion type tumor chalone and the human tumor necrosis factor to syncretize a gene vector and the expression is performed in a eukaryotic cell. The tumor chalone is combined with the AlphavBeta3 receptor on the surface of tumor tissue neovascularization endothelial cell, so that a tumor tissue vascular endothelial cell and a tumor cell can be specifically killed, and the antitumor effect and the tumor angiopoiesis inhibiting effect can be achieved.

Description

1. Technical field [0001] The present invention relates to gene cloning, gene recombination, secretion and expression of exogenous genes in eukaryotic cells in the field of medical biotechnology, and in particular to a method for preparing fusion protein tumorstatin-tumor necrosis factor by genetic engineering, including the secretion of fusion protein Type eukaryotic expression vector, the host cell containing the vector, and some biological activities of the fusion protein. 2. Background technology [0002] Traditional treatments such as chemotherapy, radiotherapy, and biological therapy target the tumor cells themselves. However, solid tumors, unlike hematological tumors, must obtain nutrients from the host through blood vessels. The formation of solid tumors depends on the formation of new blood vessels in the tumor itself, and the regeneration of tumor blood vessels is regulated by many vascular stimulation and inhibitory factors. With the rapid development of tumor a...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/62C07K19/00A61K48/00A61P35/00
Inventor 罗以勤赵亮姚丽娟
Owner 罗以勤
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