Defected slow virus vector derived from HIV-1 virus

A lentiviral vector, HIV-1 technology, applied in the direction of biochemical equipment and methods, applications, botany equipment and methods, etc., can solve the problems of narrow host range, low virus titer, poor biological safety, etc., so that it is not easy to induce , good biological safety, stable expression effect

Inactive Publication Date: 2006-07-26
南京兰卫医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The purpose of the present invention is: for the low virus titer of the first-generation lentiviral vector, the host range that can be infected is narrow and the chance of producing RCL is relatively high, and the biological safety is poor; the second-generation lentiviral vector still has no solution Practical issues of safety, providing a new defective lentiviral vector derived from HIV-1 virus

Method used

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  • Defected slow virus vector derived from HIV-1 virus
  • Defected slow virus vector derived from HIV-1 virus
  • Defected slow virus vector derived from HIV-1 virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Construction of pLenti-GFP.

[0024] (1), using the lentiviral vector pLXB isolated from the sequence of the cis-acting element (such as packaging signal, long terminal repeat sequence) and trans-acting protein in the HIV-1 genome as the original vector, it was used Xba I and Pst I digested, recovered the large fragment, digested the pHit-Sin vector with the same double restriction enzyme, obtained the 3′-LTR fragment with the U3 region removed, and connected it with T4 DNA ligase to obtain pLXB-Sin.

[0025] (2), digest pLXB-Sin with Pst I and BamH I, and reclaim the large fragment; cut the WPRE fragment from pSK-WPRE with Pst I and Sal I enzymes, and use Sal I and BamH I to cut EGFP from pEGFP-C1 Cut up and down, and then connect the above three fragments with T4 DNA ligase to obtain pLenti-GFP (structure as figure 1 ), the insertion was confirmed by restriction enzyme digestion.

Embodiment 2

[0026] Example 2. Production of pLenti-GFP lentiviral supernatant.

[0027] ①. Inoculate 10 5 293T cells were subcultured at a rate of 1:3 when the cells grew to 70-80% confluence, and DMEM (purchased from GIBCO-BRL Company) containing 10% calf serum (purchased from Hangzhou Sijiqing Company) was used as a culture medium. Cultivate for 24 hours in a 5% CO2 incubator.

[0028] ②. When the cells reach 70% confluence, transfect the pLenti-GFP plasmid lentiviral vector and the packaging plasmid with the Tat element removed by calcium phosphate co-precipitation method.

[0029] ③. Change the cell culture medium after the cells have grown for 24 hours, and culture in a 5% CO2 incubator at 37°C. Harvest the virus supernatant after 24 hours and change the cell culture medium.

[0030] ④. Harvest the virus supernatant 72 hours after transformation, and treat the cell culture dish with bleach to destroy the virus producing cells.

[0031] 5. Ultracentrifuge the virus supernatant at 5...

Embodiment 3

[0035] Identification of embodiment 3 transfection efficiency

[0036]Inoculate 10 in a 100 mm culture dish (Corning) 6 Hela cells were cultured in 10 ml of DMEM medium (purchased from GIBCO-BRL Company) containing 10% fetal bovine serum (purchased from Hangzhou Sijiqing), and subcultured at 1:3 when the cells were 80% confluent.

[0037] After 24 hours, suck out the medium, add 5ml of supernatant, culture in a 37°C incubator for 3h, and then supplement with 5ml of fresh DMEM medium; after 24h, perform pLenti-GFP lentivirus infection once in the same way, culture in a 37°C incubator for 24h, and then subculture , photographed with a fluorescence microscope. image 3 Fluorescent photo with figure 2 A comparison shows that almost 100% of cells are green cells after lentiviral vector infection, and the infection efficiency is nearly 100%.

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Abstract

The invention discloses a defective slow virus carrier form HIV-1 virus, which comprises the following steps: using cis-form action element of HIV-1 genom and slow virus carrier pLXB from sequence of reaction type protein as initial carrier; obtaining pLXB-Sin by linking up with initial carrier and pHit-Sin carrier of removed U3 district 3íõ-LTR fragment; inserting WPRE element and EGFP; obtaining pLenti-GFP. The invention possesses infected non-cleavage cell, cleavage cell and goal DNA sequence.

Description

Technical field: [0001] The invention relates to a novel lentiviral vector, especially a defective lentiviral vector derived from HIV-1 virus. Background technique: [0002] The transfection efficiency of non-viral vectors is low, and most of the gene therapy programs used in human experiments are transferred by virological methods, among which retroviral and adenoviral vectors are the most mature. Adenoviral vectors have made great achievements in gene therapy. It has high transfection efficiency, a large enough host spectrum, and can infect quiescent cells. However, the target gene is not integrated into the target cell genome and can only be expressed briefly, and the adenovirus itself Some of the antigenicity of the human body can cause an immune response to prevent its repeated transduction. [0003] Modern medical research has proved that many human difficult diseases are directly related to genes. For diseases with high morbidity and mortality such as cancer, the tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867
Inventor 孙倍成王尤山罗望许淼徐玲张泓
Owner 南京兰卫医学检验所有限公司
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