Lentiviral vector of pseudotyped lymphocytic choriomeningitis virus glycoprotein

A lymphocyte and choroid plexus technology, applied in the field of lentiviral vectors, can solve the problems of inability to achieve, loss of invasiveness of virus particles, difficulty in gene transfection, etc.

Inactive Publication Date: 2013-04-24
王剑
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

2. Also due to the instability of the envelope protein, virus particles will also lose part of their invasiveness during purification
However, functional amphitropic receptors are absent in a variety of primary human cells, such as gene therapy target cells such as hepatocytes and hematopoietic stem cells
Gene transfection in these cells is therefore relatively difficult or impossible

Method used

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  • Lentiviral vector of pseudotyped lymphocytic choriomeningitis virus glycoprotein
  • Lentiviral vector of pseudotyped lymphocytic choriomeningitis virus glycoprotein
  • Lentiviral vector of pseudotyped lymphocytic choriomeningitis virus glycoprotein

Examples

Experimental program
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Effect test

Embodiment 1

[0080] Infection of TeLCeb containing LCMV-LacZ gene transfers to target cells, neutralization of vector by concentration gradient of Anti-GP Mab and vector Use of LCMV to rescue envelope protein-negative murine leukemia virus vector: to prove whether LCMV can rescue envelope protein Negative murine leukemia virus vector, env-negative packaging cell line TELCeB was infected with LCMV WE line with an infectious multiplicity (m.o.i.: indicating the number of virions after cells were infected) of 0.01. TELCeB is derived from the human fibroblast cell line Te671 and contains gag, pol and the retroviral vector MFGnlsLacZ (G.M.Crooks et al., Blood 82 (1993) 3290-3297). After LCMV infection, the titer of LTU and LCMV multiplicity Type viruses were stained with X-gal of murine fibroblast target cells and plated (in plate-shaped units, PFU). In addition, the expression of LCMV glycoproteins in infected TELCeB can be measured by flow cytometry. The result is in. The LTU is 5x10 on the...

Embodiment 2

[0088] The products of the Gag and pol genes are required for packaging retroviral RNA import into the LCMV glycoprotein pseudotype model

[0089] Experiments were performed to confirm whether retroviral RNA alone can be packaged into LCMV or whether the products of the gag and pol genes are required to assist in the import. 293 cells and MLV293gp2 cells containing gag and pol were transfected with an MLV retroviral vector containing the neo gene (MP1N), and cell lines containing stable vector fragments were prepared by G418 selection (293MP1N and 293gp2MP1N; 293gp2MP1N cell line expressing SF23 Clones were deposited at DMSC - German Center for Microorganisms and Cells, Budapest protocol number DSM ACC2374). These cells were infected with replication-competent transfection cofactors or wild-type LCMV. The results are shown in Table 2. After infection with transfection cofactors, both cells can produce infectious vectors that transfer neomycin resistance. After infection wit...

Embodiment 3

[0094] LCMV Infection with 293gpMP1N and Stable Gene Transfer to L929 Cells by Southern Blotting MLV (LCMV) Pseudotype-Mediated Retroviral Vector Gene Transfer and Stable Integration: Resistance to G418 by a Retroviral LCMV Pseudotype Transfer, indicating that the target gene has been stably integrated into the host genome. To demonstrate that the MLV (LCMV) pseudonymous model mediates stable transduction by integrating genes into the host genome, a retroviral vector containing the neomycin repressor gene (neo) was infected by LCMV into the neo-negative enveloped cell line 293gp2MP1N to confirm. Titers were measured by shifting the inhibitory effect of G418 on Sc-1 cells in the range of 1x10 3 -1x10 4 between GTU / ml. Inhibitory cell clones emerged after 8 days and were cultured for an additional 3 weeks. Southern blot analysis of DNA from 12G418-resistant clones was performed with a neo probe after inhibition with monocutase HindIII. One gene copy of the integrated retrov...

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Abstract

Belonging to the field of molecular biological technologies, the invention relates to a lentiviral vector of pseudotyped lymphocytic choriomeningitis virus glycoprotein. The cell line provided in the invention contains a retrovirus gag gene, a pol gene, an env gene, a retroviral regulatory gene, a gp gene for encoding the glycoprotein LCMV virus GP1 and GP2. The invention first puts forward that a vector system can be manufactured under the conditions of high titer and high concentration. Vector particles can be purified without any infectious material loss. The cell line involved in the invention has a wide and cross-species host cell spectrum.

Description

technical field [0001] The invention relates to a lentiviral vector of pseudotyped lymphocytic choriomeningitis virus glycoprotein and belongs to the field of molecular biology technology. Background technique [0002] The application of retroviral vectors in new technologies is increasing, such as the fields of gene transfer in genetic engineering, medical research and gene therapy (for example: C. Baum et al. published "" by Gerson & Lattime Academic Press in 1998 Oncology Discussion: Gene Therapy for Cancer, Translational Approaches from Preclinical Research to Clinical Implementation"). Most retroviruses are derived from murine leukemia virus (MLV). They contain all the LTR region sequences necessary to maintain the integrity of the virus as well as the Ψ element responsible for the packaging function. The regions that code for viral proteins are replaced with foreign genes and control sequences that the researchers want to transfer into human cells. These vectors are...

Claims

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Application Information

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IPC IPC(8): C12N15/867A61K48/00A61P31/14
Inventor 王剑
Owner 王剑
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