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Method for cloning, identifying and expressing fusion and non-fusion genes of angiostatin Kringle 5

A technology of suppressor and fusion gene, applied in biochemical equipment and methods, botanical equipment and methods, genetic engineering, etc.

Inactive Publication Date: 2010-03-17
SHANXI LIFEGEN
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Problems solved by technology

However, the mass production of K5 will encounter some difficulties, such as how to achieve its high-efficiency expression, how to optimize its fermentation conditions and optimize the separation and purification technology of K5

Method used

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  • Method for cloning, identifying and expressing fusion and non-fusion genes of angiostatin Kringle 5
  • Method for cloning, identifying and expressing fusion and non-fusion genes of angiostatin Kringle 5
  • Method for cloning, identifying and expressing fusion and non-fusion genes of angiostatin Kringle 5

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Embodiment Construction

[0173] A method for cloning, identification and expression of angiogenesis inhibitory factor Kringle 5 fusion gene:

[0174] (1.) Purpose: To construct angiostatinK5 cDNA expression vector, and introduce it into Escherichia coli, extract and identify by enzyme digestion. Competent cells were induced to express K5 gene by small-scale fermentation, and the expressed product K5 was identified by SDS-PAGE. (2.) Method: the angiostatinK5 cDNA is contained in the plasmid of the ThioA / L15 / 7 strain, the target sequence is amplified by PCR after the plasmid is extracted, the product is purified and then digested with double enzymes, and then the target gene is connected to the vector pET32a and the recombinant vector pET32a / kringle5 Introduce the competent cells E.coli BL21, culture and extract the plasmid, and finally identify it by agarose gel electrophoresis after double enzyme digestion. For small-scale fermentation, IPTG was used to induce the expression of K5 gene in the transfo...

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Abstract

The invention relates to a method for cloning, identifying and expressing fusion and non-fusion genes of angiostatin Kringle 5. The method comprises the following steps: 1, preparation of ThioA / L15 / 7and E. coli PET32 (vector) plasmid DNA; 2, PCR of the target gene Kringle5; 3, extraction and purification of a PCR product; 4, double digestion of the PCR product and the vector plasmid DNA; 5, reclamation of DNA gel; 6, preparation of the recombinant vector pET32 / kringle5; 7, preparation of a competent cell; 8, conversion of the competent cell; 9, miniature fermentation and induction expressionof the recombinant protein; and 10, culture and plasmid extraction, and identification of agarose gel electrophoresis after double digestion. The method successfully constructs the non-fusion expression vector of the pET28a (+)-K5 gene, and establishes foundation for non-fusion expression of the K5 gene in colibacillus.

Description

technical field [0001] The invention relates to a method for cloning, identifying and expressing fusion and non-fusion genes of angiogenesis inhibitory factor Kringle 5. Background technique [0002] About 5 million people die from cancer every year in the world. [0003] As early as the 1970s, folkman proposed that tumor growth is vascular-dependent. The growth of blood vessels has two distinct phases, from the slow growth phase without blood vessels to the rapid growth phase with blood vessels. Angiogenesis, which enables tumors to obtain sufficient nutrients, is a key link in the above transformation. Angiogenesis mainly depends on the regulation of angiogenesis stimulators and inhibitors. [0004] In the field of tumor treatment, the strategy of directly killing tumor cells is dominant, that is, tumor resection or radiotherapy is performed to make the primary tumor regress, and then radiotherapy and chemotherapy are performed to eliminate the remaining cancer cells in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/70C07K14/47
Inventor 边六交杨晓燕冯萱陈超
Owner SHANXI LIFEGEN
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