Method for homologous recombining by using spirulina and expressing human gene

A technology of homologous recombination and spirulina, applied in the new field of spirulina genetic engineering, can solve problems such as difficulty in application, and achieve the effects of being suitable for consumption, high nutritional value and high growth rate

Inactive Publication Date: 2004-09-15
广东梅雁蓝藻有限公司
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Problems solved by technology

Tumor necrosis factor has a strong lethal effect on tumor cells, but it has no selectivity, that is, it also has a strong lethal effect on normal human cells, so it is difficult to apply clinically

Method used

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Embodiment Construction

[0022] Below in conjunction with embodiment, the present invention will be further described.

[0023] 1. Recombinant plasmid construction of Spirulina resistance marker gene

[0024] Spirulina is sensitive to chloramphenicol, and the chlorotoxin acetyltransferase CAT gene is suitable as a resistance screening gene for transformants of Spirulina. (1) SalI digests the plasmid containing the CAT gene expression cassette, and reclaims the CAT gene fragment; (2) EcoRI digests the plasmid containing the chloroplast photosynthetic gene expression cassette, and reclaims the photosynthetic gene promoter and terminator fragments; (3) the above Fragments obtained with T 4 After DNA ligase ligation, recombinant plasmid (I) was obtained.

[0025] 2. Construction of resistant recombinant carrier plasmid containing target gene

[0026] TRAIL is a membrane protein with one terminal sequence inserted into the membrane. The TRAIL gene required by the applicant is a functional fragment that...

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Abstract

The invention refers to a method for reconstructing the cloned and constructed human body tumor death factor TRAIL gene expression carrier into spirulina gene group to be expressed stably with gene gun. The special components of the reconstructed TRAIL expression carrier include resistant mark gene expression box CAT, chlorophyll body photosynthetic gene promotor and ender, spirulina homologous segment, TRAIL gene segment without coding film combining part, the expression system also includes characters of primary nucleus and eukarya. The invention sets suitable striking parameter, uses gene gun to guide the reconstructed expression carrier into spirulina silk body, and through several times of sifting of chloramphenicol with incremental density and molecular biology testing, the invention gets spirulina converter of TRAIL gene. The invention applies to spirulina reconstruction and human body gene expression, it also applies to reconstruct and express other primary nucleolus and eukarya.

Description

technical field [0001] The invention relates to a new technology of spirulina genetic engineering, which is used to obtain the spirulina transformants of homologous recombination and stable expression of human genes. Background technique [0002] So far, the molecular genetic background of Spirulina is still unclear. Only nearly 10 genes, such as phycocyanin and phytoene synthase, have been cloned from Spirulina at home and abroad, and little is known about the sequence and structure of the Spirulina genome. . In the absence of understanding of the genome structure and genetic background of Spirulina, it is not feasible to design a conceptual method of transforming Spirulina with exogenous genes. Researchers must construct an expression plasmid that can match, be compatible and recombined with Spirulina through a specific exogenous gene, and use effective transformation methods to create a recombination and expression system for Spirulina. This is the difficulty of Spiruli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/63
Inventor 钱凯先王勇苏中亮曾文辉李江平王宏
Owner 广东梅雁蓝藻有限公司
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