Site-specific integration retroviral vector system and preparation thereof

A lentiviral vector and site technology, applied in the field of genetic engineering, can solve problems such as hindering the application of lentiviral vector system and insertion mutation, and achieve the effect of overcoming insertion mutation, inhibiting gene therapy, and avoiding toxicity

Inactive Publication Date: 2008-11-12
北京康爱瑞浩细胞技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, like other viruses such as retroviruses, the integration of lentivirus is random, which often leads to the risk of insertion mutation, which greatly hinders the widespread application of lentiviral vector system in gene function and gene therapy

Method used

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  • Site-specific integration retroviral vector system and preparation thereof
  • Site-specific integration retroviral vector system and preparation thereof
  • Site-specific integration retroviral vector system and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation method of novel lentiviral packaging vector CMVΔ8.9-D64N / D64N

[0037] Synthetic oligonucleotide sequences D64N, F-5-TATGGCAGCTAAATTGTACACATT-3, D64N, R-5-AATGTGTACAATTTAGCTGCCATA; D116NF-5-ACAGTACATACAAACAATGGCAGC-3 and D116NR-5-GCTGCCATTGTTTGTATGTACTGT-3, using the plasmid CMVΔ8.9 as a template, using The mutation kit was used for directional mutation, enzyme digestion identification, and sequence analysis was correct. CMVΔ8.9-D64N / D116N was obtained.

Embodiment 2

[0038] Example 2 Preparation of site-specific integration lentiviral vector

[0039] Synthesize the ATTB oligonucleotide sequence 5-ATAATCGGTACCGGGTGCCAGGGCGTGCCCTTGGGCTCCCCGGGCGCGTACTCCATACTAGTTT-3 with the KpnI / speI restriction site, and clone it into the CMVC31 plasmid after annealing and recovery to obtain the CMV-ATTB-C31 plasmid. Amplify the Attb-C31 integrase coding sequence, The primers used contained the restriction site of pacI, and the PCR products were recovered after digestion.

Embodiment 3

[0040] Example 3 Preparation of site-specific integrative lentivirus packaged by plasmid CMVΔ8.9-D64N / D64N

[0041] The FattbC31UGW vector was mixed with the packaging construct plasmid CMVΔ8.9-D64N / D64N and the envelope plasmid (VSVG) at a mass ratio of 2:1:1. Use liposome LipofectamineTM to transfect on 293T cells, observe under a fluorescent microscope after 24-48 hours, collect the virus supernatant after a large amount of fluorescence appears, and collect the collected virus supernatant after concentration for equipment or use immediately. Recombinant lentivirus activity During the measurement, the concentrated virus stock solution was diluted in different proportions, and the fluorescence count was performed under a fluorescence microscope after infecting the cells for 48 hours to determine the titer. FUGW vector served as a control. The results showed that the site-specific integration lentivirus titer was 6.8*10 -7 TU / ml.

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Abstract

The invention belongs to the gene engineering field and relates to a site-specific integration slow virus vector and a method for preparing the same. The vector system consists of a FattbC31UGW plasmid, CMV delta 8.9-D64N/D116N and an envelope plasmid VSVG. To overcome the risk of insertion mutation caused by random integration of the slow virus vector system, the method remodifies the prior slow virus vector system, and obtains the site-specific integration slow virus vector system through the combination with a phiC31 integrase system. A transgene carried by the virus vector can be steadily integrated on a special site of a host genome. As the novel slow virus vector integrating effectiveness, safety and operation simplicity, on the one hand, the site-specific integration slow virus vector can be used to identify the function of an exogenous gene, in particular the function in genetically modified cell engineering; on the other hand, the method provides safer and more efficient novel approach for gene therapy in fields such as tumor inhibition and virus resistance and so on.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a site-specific integration lentiviral vector system and a preparation method thereof. Background technique [0002] Lentivirus belongs to the subgenus of retrovirus, represented by human immunodeficiency virus (human immunodeficiency virus, HIV). Lentivirus not only has the ability to infect and divide target cells and integrate into its genome, especially has the ability to infect neuronal cells, Macrophages, liver cells, cardiomyocytes, and stem cells have a variety of non-dividing cell capabilities. Therefore, lentiviral vectors have been used as effective tools for gene transfer and have been widely used in the study of gene functions, especially gene therapy. [0003] The lentiviral vector based on human immunodeficiency virus type 1 (human immunodeficiency virus, type 1; HIV-1) has been developed to the third generation. The first generation is the lentiviral vector of re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867
Inventor 朱焕章刘绍辉辛清婷余龙
Owner 北京康爱瑞浩细胞技术有限公司
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