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Safe lentivirus vector and application thereof

A technology of lentiviral vectors and safety, which is applied in the field of safe lentiviral vectors and its applications, can solve the problems of potential safety hazards of lentiviral vectors, and achieve the effects of high security, improved security, and reduced read-through rate

Active Publication Date: 2014-12-31
SHANGHAI CHILDRENS HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore, the technical problem to be solved by the present invention is to provide a highly safe lentiviral vector for the deficiency that current lentiviral vectors have potential safety hazards.

Method used

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  • Safe lentivirus vector and application thereof
  • Safe lentivirus vector and application thereof
  • Safe lentivirus vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] 1. Fragment synthesis

[0060] a) Amplification of the AHSP fragment.

[0061] Using upstream primer AHSP-F: 5'-TGGCTCTTGCCTTGCATTTCC-3' and downstream primer AHSP-R: 5'-GGTAGAGTGGCAGGAGCACAG-3', human peripheral blood cell genomic DNA was used as template for PCR amplification. The reaction system is as follows:

[0062]

[0063] A total of 4 reaction tubes were made, and the reaction conditions were 94°C for 5min; (94°C for 45sec, 56°C for 45sec, 72°C for 1min) x 32 cycles; 72°C for 10min. AHSP fragments of about 1 kb (including AHSP expression cassettes, including promoters, coding sequences, and terminators) were amplified. After mixing the reaction solution, 5 μL was sent to Shanghai Boshang Biotechnology Co., Ltd. for sequencing. Then the sequence obtained by sequencing was compared with the sequence of NC_000016.9 and found to be completely consistent.

[0064] All remaining PCR products were added to 2 times volume of ethanol precipitation, dissolved in 10...

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Abstract

The invention discloses a safe lentivirus vector and an application thereof. The lentivirus vector carries an intergrase Cre gene expression cassette, a U3 region of 3'LTR contains an LoxP (locus of X-over in P1) site sequence, and a restrictive enzyme site for introducing exogenous gene is contained is in the U3 region and is at the downstream of the LoxP site sequence. The lentivirus vector mediates the gene transfer, and a ''suicide effect'' is generated after generated pseudoviruses are integrated, so the basic skeleton of most lentiviruses is deleted, and the possibility of the generation of the recombination of a lentivirus compatibility virus (LCV) is eliminated. The readthrough rate of the lentivirus of the invention is reduced, so the safety is further improved. The safe lentivirus vector can be applied to the gene cloning or expression, especially the gene treatment. The safety of transgene mediated with the lentivirus vector of the present invention is high.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a safe lentiviral vector and its application. Background technique [0002] β-thalassemia (abbreviated as β-thalassemia) is a kind of genetic disease caused by hemoglobin (HbA) production disorder. The synthesis of βHb is inhibited during the production of HbA, which destroys the balance of the two subunits of hemoglobin, and the relatively excess αHb produces toxic deposition in red blood cells, leading to hemolytic anemia. Aiming at the pathogenesis of β-thalassemia, Wang Baobin et al. constructed an α-hemoglobin stable protein (AHSP) gene expression vector that can specifically bind to free αHb and introduced it into β654 thalassemia mice in order to prevent excess αHb from producing toxic deposition . Through molecular biology, biochemistry, pathology and hematology and other technical means, it was found that the thalassemia phenotype of mice was improved (Transgenic Humanα-He...

Claims

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Application Information

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IPC IPC(8): C12N15/867
Inventor 曾溢滔方彧聃张敬之
Owner SHANGHAI CHILDRENS HOSPITAL