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Preparation method and application of 4-1BB-containing lentivirus

A technology of lentivirus and lentiviral vector, which is applied in the field of medical biology, can solve instability and other problems, and achieve the effect of reducing procedures, costs and pollution risks

Inactive Publication Date: 2016-09-07
WUHAN SIAN MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] As the closest existing technology to this application, the patent with the publication number CN201510279570.3 discloses a lentivirus with high transfection ability and biological activity for preparing CART cells. The related sequences of CD3 and CD28 are combined with After VSVG recombination, it is used for lentivirus packaging. The first signal of CD3 and the second signal of CD28 are separately recombined with VSVG to activate T cells together. At the same time, no antibody is used to activate T cells before infecting T cells. The activation method has certain risks and instability

Method used

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  • Preparation method and application of 4-1BB-containing lentivirus
  • Preparation method and application of 4-1BB-containing lentivirus
  • Preparation method and application of 4-1BB-containing lentivirus

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Experimental program
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Effect test

Embodiment 1

[0065] A method for preparing a lentivirus comprising 4-1BB, comprising the following steps:

[0066] A. Construction of lentiviral vector plasmid 881-3-SJ19:

[0067] A1. Perform PCR amplification on the CD19CAR gene: use the whole gene synthesis CD19CAR gene as a template to perform PCR amplification to obtain the CD19CAR amplification product;

[0068] In step A1, the total volume of the PCR reaction system is 50 μL: including 0.25 μL of Ex Taq enzyme, 5 μL of Ex Taq buffer, 4 μL of dNTP, 2 μL of template and 4 μL of primers, and the balance is sterilized water. The concentration of dNTP is 2.5mM, the concentration of template is 100ng / μL, and the concentration of primer is 10uM;

[0069] The PCR amplification program used was: 25 cycles, each cycle including the following steps in turn: pre-denaturation at 98°C for 10 s, annealing at 50°C for 30 s; extension at 72°C for 2 min; After one cycle, the PCR amplification procedure is completed.

[0070] A2. Gel purification o...

Embodiment 2

[0106] Lentivirus infection of T cells and its effect detection

[0107] 1. Machine mining can obtain about 1×10 7 Mononuclear lymphocytes; mononuclear cells were isolated by Ficoll, cultured in vivo15 medium containing IL-2 (1000U / ml), and stimulated with a small amount of CD3 antibody (100ug / ml). Adjust the cell concentration to 1 × 10 before infection 6 / ml, take a volume of 1ml, culture in a 6-well plate; calculate the amount of virus according to MOI=5, add 1ml of complete culture solution to suspend before adding the virus concentrate, and filter at 0.22um. After adding the virus, mix it gently, put it into the incubator and cultivate it for 3 days, wash off the virus and continue the culture.

[0108] 2. Detection of infection efficiency

[0109] Collect 5×10 5 Cells; wash three times with buffer (PBS+2% BSA); resuspend cells in 200 μL of buffer, add 1 ug of protein L-biotin, 4°C for 45 min; wash once with buffer; resuspend cells in 200 μL of buffer, add 10 μL of S...

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Abstract

The invention provides a preparation method and application of 4-1BB-containing lentivirus. According to the lentivirus prepared by the preparation method for the lentivirus, CD28 can be not required to be added; only a small amount of CD3 is directly added to a culture medium to serve as a first T-cell activating signal before transfection, so that a process of preparing Car-T cells is reduced, and the cost and pollution risk are reduced; T-cells are stimulated to be further activated through recombinant VSVG (Vesicular Stomatitis Virus Glycoprotein), so that the contact probability of a virus and cells is improved to effectively improve infection efficiency.

Description

technical field [0001] The invention belongs to the field of medical biotechnology, and specifically relates to a preparation method and application of a lentivirus containing 4-1BB. Background technique [0002] Lentivirus vector is a gene therapy vector developed on the basis of HIV-1 (human immunodeficiency virus type I). Different from general retroviral vectors, it has the ability to infect both dividing cells and non-dividing cells. The research on lentiviral vectors is developing rapidly, and the research is also very in-depth. The vector can effectively integrate the foreign gene into the host chromosome, so as to achieve persistent expression. In terms of infection ability, it can effectively infect various types of cells such as neuron cells, liver cells, cardiomyocytes, tumor cells, endothelial cells, stem cells, etc., so as to achieve good gene therapy effects. Clinical research has been carried out in the United States. It is very ideal, so it has broad appli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/867C12N15/66C12N5/0783
CPCC07K14/005C07K14/70503C07K14/70578C12N5/0636C12N7/00C12N15/66C12N15/86C12N2740/15021C12N2740/15043C12N2760/20222
Inventor 董坚杨益刘玉芝何群周棠怡梁福蓉
Owner WUHAN SIAN MEDICAL TECH CO LTD
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