Expression plasmid for rep gene, RBE cis element fixed point integration system and its preparation method and uses

A technology of site-specific integration and cis-element, applied in the fields of biotechnology and gene therapy, can solve the problems of insertion mutation, safety concerns, etc., and achieve the effect of improving short expression time and simple preparation procedure

Inactive Publication Date: 2009-06-24
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Gene therapy has irreplaceable advantages for the treatment of genetic diseases, but gene therapy needs to modify the patient's genome, which makes people worry about its safety
[0003] There are currently several gene manipulation transfer pathways. Viral vectors have high gene transfer efficiency, but there are many deficiencies in safety: such as adenovirus, adeno-associated virus, retroviral vectors, and lentiviral vectors, which can be randomly integrated into the host genome causing insertion mutation
However, the recombinant AAV vector constructed with ITR as a cis-element cooperates with the Rep protein, even if it can be integrated into the host body, when the host is re-infected by the co-virus, a virus carrying the foreign gene with replication ability will be produced in the body, constituting Potentially dangerous (Linden, R.M., E. Winocour, and K.I. Berns. 1996. The recombination signals for adeno-associated virus site-specific integration. Proc. Natl. Acad. Sci. USA 93: 7966-7972)

Method used

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  • Expression plasmid for rep gene, RBE cis element fixed point integration system and its preparation method and uses
  • Expression plasmid for rep gene, RBE cis element fixed point integration system and its preparation method and uses
  • Expression plasmid for rep gene, RBE cis element fixed point integration system and its preparation method and uses

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Embodiment Construction

[0024] Materials and methods

[0025] 1. Restriction enzymes AseI, BamHI, T4 DNA ligase, and klenow DNA polymerase are products of British Biolabs.

[0026] 2. Cloning and sequencing The vector pEGFPN2 is a product of Clontech Company.

[0027] 3. Promega plasmid extraction kit is a product of Promega Company. Biotech Gum Recovery Kit is a product of Biotech.

[0028] 4. The positive and negative chain nucleotide base fragments of the designed RBE cis-element were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

[0029] The above materials are commercially available

[0030] 1. Design cloning and sequencing of RBE cis-element and construction of pRBE-CMV-EGFP recombinant plasmid

[0031]Add an Asel sticky enzyme cleavage site at the 5' end of the positive strand of the RBE sequence, and add an Asel sticky enzyme cleavage site at the 3' end of the negative strand. The nucleotide base fragments of the positive and negative strands of the RBE cis-element were synthes...

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Abstract

The invention belongs to the technical field of biotechnology and gene therapy, and specifically relates to a rep gene expression plasmid, an RBE cis-element fixed-point integration system, a preparation method thereof, and an application in gene therapy. The site-specific integration system is composed of an expression plasmid carrying an RBE cis-element and an EGFP reporter gene and a rep gene expression plasmid, or an expression plasmid carrying an RBE cis-element and human coagulation factor IX and a rep gene expression plasmid. role composition. The site-specific integration system can be used to introduce foreign genes into human cell lines, has a simple preparation procedure, can be used in combination with other therapeutic genes, and can be applied to gene therapy for various diseases.

Description

technical field [0001] The invention belongs to the technical field of biotechnology and gene therapy, and specifically relates to a rep gene expression plasmid, an RBE cis-element fixed-point integration system, a preparation method thereof, and an application in gene therapy. Background technique [0002] Gene therapy has advantages that cannot be replaced by other methods for the treatment of genetic diseases, but gene therapy needs to modify the patient's genome, which makes people worry about its safety. [0003] There are currently several gene manipulation transfer pathways. Viral vectors have high gene transfer efficiency, but there are many deficiencies in safety: such as adenovirus, adeno-associated virus, retroviral vectors, and lentiviral vectors, which can be randomly integrated into the host genome causing insertion mutation. The gene transfer efficiency of non-viral vectors is low, the expression of foreign genes is short-lived, and there are also safety prob...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/11C12N15/70C12N15/66C12N1/21A61K48/00
Inventor 冯登敏朱焕章陈金中贾韦国薛京伦
Owner FUDAN UNIV
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