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Construction of secretory coenosarcus plasmid for bombyx mori rhabditis viral expression system

A silkworm baculovirus and expression system technology, which is applied in the field of construction of secretory donor plasmids, can solve the problems of recombinant expression that cannot be secreted, and achieve the effect of strong protein secretion ability

Inactive Publication Date: 2006-08-16
ZHEJIANG UNIV
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Problems solved by technology

The donor plasmid is an important tool to determine how the target gene is expressed. At present, Invitrogen in the United States has sold a product named pFastBacHTa, b, c (catalogue number: 10584-027), but the plasmid does not contain protein signal peptide secretion signal, resulting in the expression of recombinant expression can not be secreted out of the cell

Method used

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  • Construction of secretory coenosarcus plasmid for bombyx mori rhabditis viral expression system
  • Construction of secretory coenosarcus plasmid for bombyx mori rhabditis viral expression system
  • Construction of secretory coenosarcus plasmid for bombyx mori rhabditis viral expression system

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Embodiment Construction

[0012] In order to enable the recombinant protein to be efficiently secreted out of the cell, a donor plasmid with strong secretion ability was constructed. Baculovirus transfer plasmid pAcGP67-B (BD Biosciences: catalog number: 21223P, figure 1 ) has a strong secretory signal peptide gp67 sequence in the downstream of the polyhedron promoter Ph, which endows the recombinant protein with a strong ability to secrete extracellularly, and pAcGP67-B plasmid is secreted and transformed into human Escherichia coli DH5 competent cells, followed by plasmid Extract, and perform EcoRV and HindIII double enzyme digestion treatment, isolate and obtain a gene fragment with Ph promoter, gp67 signal peptide sequence and multiple cloning site, the size is 377bp; and the existing donor plasmid pFastBacHTa, b, c (U.S. Invitrogen company product, catalog number: 10584-027, figure 2 ) for SnaBI and HindIII enzyme digestion treatment, excise the original Ph promoter and its multiple cloning site...

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Abstract

The invention opened a constructing method of the secretory donor plasmid for the Bombyx Baculovirus expression system. It is based on the donor plasmid of pFastBacHTa,b,c and the Baculovirus transforming carrier pAcGP67-B. The plasmid provided the condition for getting the recombined protein.

Description

technical field [0001] The invention relates to genetic engineering technology, a baculovirus gene expression system, protein purification and activity analysis, in particular to a method for constructing a secretory donor plasmid of the silkworm baculovirus expression system. Background technique [0002] The silkworm baculovirus gene expression system is one of the most efficient eukaryotic expression systems at present, but the current technology has the disadvantages of low recombination rate, cumbersome plaque analysis technology and long time-consuming in the process of recombinant virus construction and purification. In order to better solve this problem, we learned from the working principle of the foreign AcNPV rapid gene expression system, and used the gene positioning and transfer function of bacterial transposons to realize gene transfer and recombination in E. Featured resources Insect-Bombyx mori rapid gene expression system (Patent authorized, name: Method for...

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Application Information

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IPC IPC(8): C12N15/866
Inventor 吴小锋姚慧鹏王冰
Owner ZHEJIANG UNIV
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