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Shark angiogenesis inhibiting factor and production and purification method and application

An angiogenesis inhibition and angiogenesis technology, applied in the field of bioengineering, can solve the problems of inability to large-scale clinical application and few sources.

Inactive Publication Date: 2010-11-24
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to obtain the amino acid sequence of a shark angiogenesis inhibitor by means of genetic engineering according to the problems of few sources and inability to be applied clinically on a large scale in the existing research on shark angiogenesis inhibitors

Method used

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  • Shark angiogenesis inhibiting factor and production and purification method and application
  • Shark angiogenesis inhibiting factor and production and purification method and application
  • Shark angiogenesis inhibiting factor and production and purification method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Obtaining the full-length cDNA sequence of shark angiogenesis inhibitory factor and its encoded amino acid sequence

[0037]In this embodiment, the full-length cDNA sequence of shark angiogenesis inhibitory factor (SAIF) and its encoded amino acid sequence are prepared from the cartilage tissue of the striped bamboo shark by gene cloning technology, and the specific steps are as follows:

[0038] 1. RNA Extraction and First-Strand cDNA Synthesis

[0039] The extraction and purification of total RNA extracted from the cartilage tissue of Bamboo Shark were carried out according to the instructions of the SVTotal RNA Isolation System kit from Promega Company.

[0040] The first strand of cDNA was synthesized using the TaKaRa RNA LA PCR TM Kit (AMV) kit, using the above-mentioned bamboo shark cartilage tissue RNA as a template, and oligo(dT)18 as a reverse transcription primer, and the operation was carried out according to the method recommended by the kit. Stor...

Embodiment 2

[0059] Example 2 Construction, Identification, Expression and Purification of Recombinant Vectors

[0060] 1. Construction and identification of recombinant vectors

[0061] 1.1 Design upstream and downstream primers Fw and Rv according to the gene sequence of the open reading frame in cDNA:

[0062] P1, the nucleotide sequence of which is shown in SEQ ID NO: 10;

[0063] P2, its nucleotide sequence is shown in SEQ ID NO:11.

[0064] There is an NdeI restriction site on primer P1, and a BamHI restriction site on primer P2.

[0065] 1.2PCR amplification

[0066] The reaction conditions of PCR amplification were: pre-denaturation at 94°C for 3min, 60s at 94°C, 60s at 40°C, and 60s at 72°C for a total of 30 cycles, and finally extension at 72°C for 5min.

[0067] The PCR amplification system is

[0068] PCR Buffer 4

[0069] dNTP 2

[0070] rTaq enzyme 0.2

[0071] Plasmid 2

[0072] Fw 2

[0073] Rv 2

[0074] ddH2O 7.8

[0075]

[0076] ...

Embodiment 3

[0093]Example 3 Inhibitory Effect of Recombinant Protein SAIF on Vascular Endothelial Cells and Detection of Inhibitory Growth of Chicken Embryo Allantoic Membrane Vessels

[0094] 1. Detection of recombinant protein SCAIF inhibiting proliferation of vascular endothelial cells:

[0095] Human umbilical vein endothelial cells HUVEC were used in 1×10 4 The density of cells / well was cultured in a 96-well plate, and the culture medium was Hams F-12.

[0096] The recombinant protein SAIF was added to the culture medium at concentrations of 5ug / mL, 10ug / mL, 20ug / mL, 40ug / mL, and 80ug / mL for three days, and then MTT staining was performed to observe the staining results of the recombinant protein SCAIF at different concentrations.

[0097] from Image 6 It can be seen that the recombinant protein SCAIF has an inhibitory effect on the proliferation of vascular endothelial cells in a concentration-dependent manner.

[0098] 2. Inhibition of angiogenesis in chick chorioallantoic memb...

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Abstract

The invention discloses a shark cartilage angiogenesis inhibiting factor and a production and purification method and an application. The amino acid sequence of the shark cartilage angiogenesis inhibiting factor of the invention is shown as SEQ ID NO:1, and the cDNA sequence coding the amino acid sequence of the shark cartilage angiogenesis inhibiting factor is shown as SEQ ID NO:2. The shark cartilage angiogenesis inhibiting factor has the effects of inhibiting the growth of vascular endothelial cells and the blood vessel, and can be applied to the preparation of medicines for inhibiting the angiogenesis or vessel growth, in particular the tumor angiogenesis or tumor vessel growth.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a shark angiogenesis inhibitory factor, its production and purification method and application. Background technique [0002] Sharks are one of the marine animals that rarely suffer from malignant tumors, and the incidence rate is one in a million. American scientist Luer injected a high dose of aflatoxin B1, a strong carcinogen, into sharks, but it could not induce cancer in sharks. Inoculating sharks with cancer cells also failed to induce cancer. This suggests that sharks have a unique anti-tumor mechanism (He Lihong 1, Huang Jianhua 2, Shen Songdong et al., Marine Science / 2005 / Volume 29 / Period 11: 63-66). [0003] Over the years, researchers from all over the world have mostly studied shark cartilage tissue, and isolated and extracted a variety of substances that have tumor suppressive effects, especially inhibiting the growth of tumor angiogenesis, such as Lee et al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/475C07K1/22C12N15/12C12N15/63C12N1/21A61K38/18A61P35/00C12R1/19
Inventor 谢秋玲梁旭方陈小佳洪岸
Owner JINAN UNIVERSITY
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