Recombinant slow virus vector, recombinant slow virus and stem cell containing recombinant slow virus

A technology of recombinant lentivirus and lentiviral vector, applied in the field of medical molecular biology, can solve problems such as easy malignant transformation

Inactive Publication Date: 2011-08-17
ARMY MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the problem that adult stem cells ASC are prone to malignant transformation during in vitro expansion or after transplantation, the present invention provides a recombina

Method used

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  • Recombinant slow virus vector, recombinant slow virus and stem cell containing recombinant slow virus
  • Recombinant slow virus vector, recombinant slow virus and stem cell containing recombinant slow virus
  • Recombinant slow virus vector, recombinant slow virus and stem cell containing recombinant slow virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Construction of recombinant lentiviral vector.

[0070] Sources of biological materials involved in the present invention:

[0071] T-apoptin carrier: a gift from researcher Yang Bing of Beijing Academy of Agriculture and Forestry, commercially available

[0072] FG-12: Gift from Prof. David Baltimore (David Geffen School of Medicine, University of California, Los Angeles, USA). The vector lacks E1 and E3 regions, and can insert large fragments of foreign genes. In addition to the above advantages, it also comes with an EGFP tracer gene, which can be used to trace the distribution of transplanted stem cells in vivo. See Figure 14 .

[0073] Auxiliary packaging plasmid: Invitrogen's Virapower system.

[0074] 293FT cells: purchased from Shanghai Institute of Cell Biology.

[0075] The above biological materials are preserved in this laboratory and can be released to the public for verification experiments.

[0076] (1) Survivin promoter amplification: pr...

Embodiment 2

[0100] Example 2: Comparison of three kinds of promoter activation activities (antibody detection of target protein expression)

[0101] Materials: Three kinds of recombinant lentivirus supernatant Lenti-FG-161pSurAH, Lenti-FG-272pSurAH, Lenti-FG-990pSurAH obtained in Example 1, BIO-RAD electrophoresis instrument and electroporation instrument (BIO-RAD Company, USA), primary antibody Mouse anti-His antibody (Beyond Company), secondary antibody is horseradish peroxidase-labeled goat anti-mouse IgG (H+L) (Beyond Company), ECL luminescence kit (Beyond Company)

[0102] method:

[0103] 1. Introduce 293FT into a 24-well plate, and control the quantity to ensure that the cell density is about 70-80% the next day.

[0104] 2. Using Lip200 as a medium, transfect three plasmids, FG-161pSur-ApoH-pA, FG-272pSur-ApoH-pA and FG-990pSur-ApoH-pA, according to routine operations, and the amount of transfected plasmid per well is 800ng. Plasmid (ug): Lip2000 (ul) = 1: 1.5, and a negative co...

Embodiment 3

[0113] Example 3: Recombinant lentivirus Lenti-FG-272pSurAH infects Hela cells and 10T1 / 2 cells to induce cell death

[0114] Materials: Use the recombinant Lenti-FG-272pSurAH lentiviral supernatant obtained in Example 1, Olympus 1X71-FL fluorescence inverted microscope, Hela cells, 10T1 / 2 cells

[0115] Method: The recombinant lentiviral supernatant Lenti-FG-272pSurAH obtained in Example 1 was used to infect the tumor cell line Hela cells and the mesenchymal stem cell line 10T1 / 2 cells for 72 hours.

[0116] A. Fluorescence microscope observation

[0117] 1. The tumor cell line Hela cells and the mesenchymal stem cell line 10T1 / 2 cells were grown on coverslips for 24 hours.

[0118] 2. Infect tumor cells with the recombinant lentivirus supernatant Lenti-FG-272pSurAH obtained in Example 1, and continue culturing for 72 hours.

[0119] 3. The cells were washed twice with PBS.

[0120] 4. Add 5ul 7-AAD staining solution to 500ul Binding Buffer, mix well, and add to the above-...

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Abstract

The invention discloses a recombinant slow virus vector, a recombinant slow virus and a stem cell containing recombinant slow virus, belonging to the field of medical molecular biology. The recombinant slow virus vector is characterized in that: a Survivin prompter, an Apoptin gene and a SV40Poly A sequence are sequentially inserted into a plurality of cloning sites of the slow virus vector; and the slow virus vector is FG-12. Due to the application of the recombinant slow virus, such as transfection applied to a transplanted stem cell, the stem cell transplanted into the body of a patient is under the monitoring of the promoter of the recombinant slow virus vector. Once the transplanted stem cell is transformed severely, the expression of a suicide gene Apoptin in the recombinant slow virus is started immediately, so that the severely-transformed cell is dead by apoptosis. During clinical application, the recombinant slow virus plays a role in specifically pre-warning, reacting, tracing and clearing the transformed stem cell.

Description

technical field [0001] The present invention relates to the field of medical molecular biology, in particular to a recombinant lentiviral vector and a recombinant lentivirus containing survivin (Survivin) promoters of different lengths driving apoptin (Apoptin) capable of inducing malignant transformed cells to actively undergo apoptosis, and a recombinant lentivirus containing Stem cells of the recombinant lentivirus. Background technique [0002] Although embryonic stem cells have multidirectional differentiation potential, there are problems such as therapeutic cloning technology, medical ethics, graft rejection and tumorigenicity, which make more research turn to the basic and clinical aspects of adult stem cells (ASC). related issues. ASC exists in various organs and tissues, can differentiate into various tissue cells, and has low immunogenicity and expandability. Or as seed cells for tissue engineering, or carrier cells for gene therapy, or both, ASC transplantation...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N7/01C12N5/10
Inventor 邹仲敏钟波叶枫庞学利粟永萍
Owner ARMY MEDICAL UNIV
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