Dual-host recombination rhabdovirus expression vector and construction method and application thereof

A technology of recombinant baculovirus and expression vector, which is applied in the fields of application, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of no use value, etc., and achieve the goal of reducing labor workload, reducing costs, and saving resources Effect

Inactive Publication Date: 2010-06-09
HENAN AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Recombinant baculoviruses used to express foreign genes on insect cells express foreign proteins, and the recombinant baculoviruses also proliferate in large quantities to produce high-gradient baculoviruses. Except for a part of these recombinant baculoviruses, they are used again Except for seed poisoning, most of them have no use value

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  • Dual-host recombination rhabdovirus expression vector and construction method and application thereof
  • Dual-host recombination rhabdovirus expression vector and construction method and application thereof
  • Dual-host recombination rhabdovirus expression vector and construction method and application thereof

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Embodiment 1

[0027] A dual-host recombinant baculovirus expression vector, the recombinant baculovirus expression vector simultaneously contains a Polyhedrin promoter that can be recognized by insect cells and a CMV-IE promoter that can be recognized by animal cells.

[0028] The double host recombinant baculovirus expression vector construction method, its steps are as follows:

[0029] (1) Construction of dual-host recombinant baculovirus transfer vector:

[0030] (A) Based on the transfer vector pFastBac1 series plasmids in the baculovirus Bac-to-Bac expression system, the Bst1107I restriction site was selected upstream of the Polyhedrin promoter, and inserted under the control of the Polyhedrin promoter to express in insect cells The source gene is the porcine IFN-γ sequence, and the front end of the inserted foreign gene can be introduced into a honeybee melittin signal peptide (HBM) gene that can achieve secreted expression.

[0031] (a) Design two primers to amplify the gene encodi...

Embodiment 2

[0079] A dual-host recombinant baculovirus expression vector, the recombinant baculovirus expression vector contains a Polyhedrin promoter and a p10 promoter that can be recognized by insect cells, and has a CMV-IE promoter that can be recognized by animal cells.

[0080] The double host recombinant baculovirus expression vector construction method, its steps are as follows:

[0081] (1) Construction of dual-host recombinant baculovirus transfer vector:

[0082] (A) Based on the transfer vector pFastBacDual plasmid in the baculovirus Bac-to-Bac expression system, under the control of the Polyhedrin promoter and the p10 promoter, the foreign gene sequence porcine IFN-α( Porcine interferon alpha, PoIFN-α) and IFN-γ (Porcine interferon alpha, PoIFN-γ), and at the same time, a honeybee melittin signal peptide (HBM) gene sequence that can realize secretory expression was introduced at the front end.

[0083] (a) Design primers to amplify the gene encoding melitin signal peptide by...

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Abstract

The invention discloses a dual-host recombination rhabdovirus expression vector and a construction method and application thereof, belonging to the technical field of gene engineering. The dual-host recombination rhabdovirus expression vector is provided with a recognizable Polyhedrin promoter of an insect cell and a recognizable CMV-IE promoter of an animal cell. Dual-host recombination rhabdovirus can be used for preparing a functional protein on the insect cell or can express a target foreign protein on the animal cell and can be used for gene therapy and nonreplication vector vaccine. The construction method of the dual-host recombination rhabdovirus expression vector comprises the following steps of: taking a transfer vector pFas in a Bac-to-Bac expression system as a base; introducing a CMV-IE promoter expression kit on the upstream of the Polyhedrin promoter; constructing the transfer vector; transforming a DH10Bac competent cell; recombining a dual-host Bacmid shuttle vector; and then transfecting insect cells in a logarithmic phase to obtain the dual-host recombination rhabdovirus expression vector.

Description

technical field [0001] The invention relates to genetic engineering technology, insect baculovirus gene expression system, gene therapy and live vector vaccine vector, in particular to a dual-host recombinant baculovirus expression vector and its construction method and application. Background technique [0002] The insect baculovirus gene expression system has super expression efficiency; it has the ability to post-translationally process and modify the expression product; it can maintain the spatial structure necessary for the exogenous protein to exert its biological activity; it cannot replicate in animal cells and is biologically safe. High; basically no toxicity to cells; simple operation, easy preparation; large capacity for inserting foreign genes and other characteristics and advantages, it is currently one of the most widely used eukaryotic expression systems. [0003] Invitrogen Corporation of the United States has launched the Bac-to-Bac expression system. The s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866A61K48/00
Inventor 王彦彬崔保安陈红英张红英王亚宾李新生魏占勇胡慧
Owner HENAN AGRICULTURAL UNIVERSITY
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