Foreign gene-carrying recombinant virus vector efficiently produced in packaging cell and construction method and application thereof

A technology of recombinant virus vectors and exogenous genes, applied in the field of recombinant virus vectors, can solve the problems of inability to obtain virus titers, shorten the timing of transcription and translation of each functional gene of recombinant viruses, and reduce the production efficiency of recombinant viruses, etc.

Active Publication Date: 2013-06-19
HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, on the one hand, the overexpressed exogenous genes have certain toxicity to the cells, especially the suicide genes, apoptosis genes, cell cycle-related genes, etc., which lead to the premature death of the virus packaging cells and shorten the function of each functional gene of the recombinant virus in the packaging cells. On the other hand, a large amount of transcription and translation of exogenous genes will also occupy too many enzymes and resources related to RNA transcription and protein translation in cells, thereby indirectly inhibiting Transcription and translation of functional genes of recombinant virus
The effects of two aspects reduce the production efficiency of recombinant virus in packaging cells and cannot obtain sufficient virus titers

Method used

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  • Foreign gene-carrying recombinant virus vector efficiently produced in packaging cell and construction method and application thereof
  • Foreign gene-carrying recombinant virus vector efficiently produced in packaging cell and construction method and application thereof
  • Foreign gene-carrying recombinant virus vector efficiently produced in packaging cell and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. Example 1: Construction of a recombinant adenoviral vector that carries the Rituxan gene and can be efficiently produced in 293 cells

[0043] 1. Construction of a replication-defective adenoviral vector carrying the Rituxan gene

[0044] The pDC315 vector was purchased from Microbix Biosystem Inc. (Toronto), Canada. It contains a 1417-2344bp sequence fragment of the E1 region of the left arm of adenovirus type 5 and an inverted terminal repeat (ITR), which can be identical to the backbone plasmid containing the right arm of the adenovirus. Source recombination to obtain replication-deficient adenovirus. The pDC315 vector was transformed by enzyme digestion, replaced the mCMV promoter, connected the EF1α (eukaryotic translation elongation factor 1 alpha) promoter and intron, and a new multiple cloning site, and retained the original SV40 PolyA signal sequence. The vector was named pDC338 . Polymerase chain reaction (PCR) technology was used to amplify the light cha...

Embodiment 2

[0132] 2. Example 2: Construction of a recombinant lentivirus that carries the human P53 gene and can be efficiently produced in 293FT cells

[0133] 1. Construction of expression plasmids for lentiviral packaging comprising human P53 gene

[0134] The pKCDNA-CMV--EF1α-GFP plasmid was purchased from Kangcheng Biological Company. It contains the expression cassette of the GFP gene under the control of the EF1α promoter, and the foreign gene can be expressed under the control of the CMV promoter. Human total RNA was extracted, single-strand cDNA was synthesized by reverse transcriptase, and the open reading frame of human P53 gene was amplified from the single-strand cDNA library by polymerase chain reaction (PCR). Named pKCDNA-P53.

[0135] Primer 5: CCG TCTAGA ACCATGGAGGAGCCGCAGTCAGA (SEQ ID NO: 7)

[0136] Primer 6: TGC GAATTC TCAGTCTGAGTCAGGCCC (SEQ ID NO: 8)

[0137] Primer 5 and primer 6 amplified to obtain the 1197bp coding region sequence of the P 53 gene, and Xba...

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Abstract

The invention provides a foreign gene-carrying recombinant virus vector efficiently produced in a packaging cell and a construction method and application thereof. The foreign gene-carrying recombinant virus vector is characterized in that at least one target point nucleotide sequence of the following micro RNA is inserted into the 3' non-translational zone of a foreign gene connected with a virus in an operable way; and the micro RNA expresses at a high level in a virus packaging cell, and does not express or expresses at a low level in a target cell. Therefore, the invention realizes the selective inhibition of the expression of the foreign gene in the virus packaging cell, avoids the negative effect of the foreign gene on the virus packaging and production, and improves the production efficiency and titer of the virus. Simultaneously, the invention can not lower the expression level of the foreign gene in the target cell, and can not affect the therapeutic effect or the research onthe function of the foreign gene.

Description

field of invention [0001] The present invention generally relates to recombinant viral vectors. More specifically, the present invention relates to a class of recombinant virus vectors carrying exogenous genes that can be efficiently produced in packaging cells, their construction methods and their uses. Background technique [0002] Viruses have a high infection rate for specific cells, and can efficiently transduce foreign genes into cells and express them for a long time. They are often used as a tool for gene transfer to mediate the overexpression of foreign genes in target cells, through function acquisition ( gain-of-function) to study the function of genes. On the other hand, viruses are also the most commonly used carriers for gene therapy. As of September 2008, 1,472 gene therapy programs have been used in clinical trials in the world, of which 1,006 clinical trials use different types of viruses as carriers, accounting for 10% of the total. 68.3% (see database ht...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/86C12R1/93
Inventor 钱其军金华君李琳芳吕赛群吴孟超
Owner HEPATOBILIARY SURGERY HOSPITAL SECOND MILITARY MEDICAL UNIV
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