Gene clone and expression of RGD die body protein of oral gland in Japan lamprey possessing function for anti tumour

Abstract

The gene clone and expression of Japonese lamprey's oral gland model proteins RGD with antineoplastic action are disclosed. Three cDNA sequences with said model proteins RGD and their clones, the protein sequence and its expression in engineered bacteria, and the action of proteins RGD in closing the signal transfer channel to suppress the vascular neogenesis and tumor cell reproduction for antineoplastic purpose are also disclosed.

Description

technical field

[0001] The gene cloning and expression of the RGD motif protein of the Japanese lamprey oral gland belongs to the field of biotechnology, and involves the cDNA sequences and cloning of three RGD motif proteins in the Japanese lamprey oral gland, the corresponding protein sequences and their Expression in Escherichia coli or Pichia pastoris, and the three recombinant proteins block the integrin-mediated signal transduction pathway by antagonizing the binding of certain integrins highly expressed on the surface of neovascular endothelial cells and tumor cells to the extracellular matrix , thereby inhibiting angiogenesis and inhibiting the proliferation, adhesion, infiltration and metastasis of tumor cells, and causing the apoptosis of vascular endothelial cells and tumor cells, so as to achieve anti-tumor effects, and can be used in the preparation of angiogenesis inhibitors anti-tumor drugs in the application. Background technique

[0002] The Japanese lampre...

Examples

Embodiment Construction

[0062] 1. Extraction of total RNA: Trizol reagent of GIBCOBRL was used. details as follows:

[0063] (1) Take the oral gland of Japanese lamprey and quickly place it in liquid nitrogen for preservation;

[0064] (2) Weigh 0.2 g of oral gland tissue, add 1 ml TRIzol reagent to prepare toxic gland homogenate, and incubate at 4°C for 5 min;

[0065] (3) Add 0.2ml chloroform, close the lid tightly, shake vigorously for 15sec, and then place it on ice for 5min;

[0066] (4) 4°C, 12000xg, centrifuge for 15 minutes;

[0067] (5) Transfer the upper aqueous phase to another centrifuge tube, add 0.5ml of isopropanol, and incubate on ice for 10 minutes;

[0068] (6) 4°C, 12000xg, centrifuge for 15 minutes;

[0069] (7) Discard the supernatant, add 1ml of 75% ethanol to the precipitate (containing RNA) to wash, and vortex to mix;

[0070] (8) Centrifuge at 10,000xg for 5 minutes at 4°C to obtain RNA precipitates;

[0071] (9) After air drying, dissolve with appropriate amount of TE ...

technical field

[0001] The gene cloning and expression of the RGD motif protein of the Japanese lamprey oral gland belongs to the field of biotechnology, and involves the cDNA sequences and cloning of three RGD motif proteins in the Japanese lamprey oral gland, the corresponding protein sequences and their Expression in Escherichia coli or Pichia pastoris, and the three recombinant proteins block the integrin-mediated signal transduction pathway by antagonizing the binding of certain integrins highly expressed on the surface of neovascular endothelial cells and tumor cells to the extracellular matrix , thereby inhibiting angiogenesis and inhibiting the proliferation, adhesion, infiltration and metastasis of tumor cells, and causing the apoptosis of vascular endothelial cells and tumor cells, so as to achieve anti-tumor effects, and can be used in the preparation of angiogenesis inhibitors anti-tumor drugs in the application. Background technique

[0002] The Japanese lampre...

Examples

Embodiment Construction

[0062] 1. Extraction of total RNA: Trizol reagent of GIBCOBRL was used. details as follows:

[0063] (1) Take the oral gland of Japanese lamprey and quickly place it in liquid nitrogen for preservation;

[0064] (2) Weigh 0.2 g of oral gland tissue, add 1 ml TRIzol reagent to prepare toxic gland homogenate, and incubate at 4°C for 5 min;

[0065] (3) Add 0.2ml chloroform, close the lid tightly, shake vigorously for 15sec, and then place it on ice for 5min;

[0066] (4) 4°C, 12000xg, centrifuge for 15 minutes;

[0067] (5) Transfer the upper aqueous phase to another centrifuge tube, add 0.5ml of isopropanol, and incubate on ice for 10 minutes;

[0068] (6) 4°C, 12000xg, centrifuge for 15 minutes;

[0069] (7) Discard the supernatant, add 1ml of 75% ethanol to the precipitate (containing RNA) to wash, and vortex to mix;

[0070] (8) Centrifuge at 10,000xg for 5 minutes at 4°C to obtain RNA precipitates;

[0071] (9) After air drying, dissolve with appropriate amount of TE ...

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