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Construction of CD3 specific lentivirus and application thereof

A recombinant virus, l1-z1-scfv-z2-tm-z3 technology, applied in the field of medical biology, can solve the problems of complicated and cumbersome process, difficult to popularize CAR-T cell therapy, and long preparation time.

Inactive Publication Date: 2021-06-01
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The process of CAR-T cell therapy is complex and cumbersome, and the preparation takes a long time, making CAR-T cell therapy difficult to popularize and expensive

Method used

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  • Construction of CD3 specific lentivirus and application thereof
  • Construction of CD3 specific lentivirus and application thereof
  • Construction of CD3 specific lentivirus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0236]Example 1 CD3 specific transformation of a slow viral envelope protein (plasmid structurefigure 1 Indicated by)

[0237]1.1 Synthesis of sequence of hemorrhoids and associated primers

[0238]The nucleic acid sequence of leprosy virus hemagodile protein sends a living organism. Design-related primers are shown in Table 1, allowing it to add appropriate Linker between H protein and OKT3 during PCR.4S)3. Synthetic primer 4000rpm Centrifuge 5min to add the appropriate amount of DDH2O, the primer is finally concentrated to 10 um.

[0239]Table 1. Another primer, sequencing primers and identification primers for the construction of plasmids

[0240]

[0241]1.2 Double-CMV-VSVG (SalI, BglI)

[0242]After the plasmid P-CMV-VSVG is incubated with restriction endonuclease SALI, BGLI, the electrophoresis cream product, the electrophoresis of gel imaging resultsfigure 2 The colloidal recovery linear carrier P-CMV-VSVG (SALI, BGLII) product, size of about 4063 bp (base pair).

[0243]1.3 PCR amplification of ...

Embodiment 2

[0268]Example 2 Packaging Production Slow Virus

[0269]2.1 293FT cell culture

[0270]293FT cells are provided by Fujian Medical University Immunotherapy Research Institute, and the recovery in liquid nitrogen tanks. After washing to wash away the freeze-saving solution, add about 5 ml of DMEM complete medium, and take about 2 × 10 after counting.6Cell293FT cells in 75cm2About 10 ml of DMEM complete medium is added to the culture bottle, CO2Culture of constant temperature incubator.

[0271]2.2 Slow Virus PCDH-GFP-VSVG and PCDH-GFP-HMO Packaging Production

[0272]One day before transfection, 293FT cells in digestive state, 6-well plate 4-hole cells, according to 4 × 105Cell / hole average holes, 37 ° C CO2Culture of constant temperature incubator. The second day of the microscope views the cells, and the transfection can be carried out at between 70% -90%. DMEM was completely cultured with DMEM before transfection. Take 2 EP tubes, labeled VSVG groups (positive controls), and the amount of pl...

Embodiment 3

[0280]Example 3 Slow viral infection

[0281]3.1 Choose a suitable cell

[0282]293FT cells and Jurkat cells were selected for experiments, 293FT cells and Jurkat cells were provided by Fujian Medical University Immunology Institute. 293FT cells are human embryonic renal cells and does not express CD3. Jurkat cells are human peripheral blood hyzogeneous T cells, stably expressing CD3.

[0283]3.2 Slow virus infection 293FT / jurkat cells

[0284]Ten wells with a good state of 293FT and Jurkat cells, and count the 9 holes of the 24-well plates. Add a virus stock solution infection to the experimental group cells (see Table 5 for details).

[0285]Table 5. Each experimental group slow virus stock solution when infected with 293 ft / jurkat cells

[0286] cell VSVG group (UL) HMO group (UL) Blank control 293FT 7575Unable Jurkat7575Unable

[0287]The blank control group added isometric DMEM complete medium / 1640 fully medium, sealing film seal 24-well plate, 2000 rpm, 37 ° C centrifugation 90mins, re...

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Abstract

The invention provides construction of a CD3 specific lentivirus and application of the CD3 specific lentivirus. Specifically, the invention provides a fusion protein, and a virus vector containing the fusion protein or a recombinant virus . The CD3 specific lentivirus disclosed by the invention can be used for efficiently transfecting mammalian cells.

Description

Technical field[0001]The present invention belongs to the field of medical biotechnology, and in particular, the present invention relates to the construction of a CD3 specific lentivirus and its application.Background technique[0002]Today's cancer is in high levels, and patients with cancer are increasing. If there is no corresponding measure, it is expected that by 2020, the annual addition of cancer will reach 15 million people. CAR-T cells (Chimeric antigen receptor tcells, car-t cells) therapy as a new tumor treatment strategy focused. Especially in the treatment such as refractory pre-b cell acute leukemia, a great breakthrough in blood system diseases such as Diffuse Large B cell Lymphoma, various The treatment and research in solid tumors is also progressive. Novartis company's KymriahTM And Gilead YescartaTM Since 2017, in October and October, Food and Drugadministration (FDA), approved to children's acute lymphocyte leukemia (ALL) and adult diffuse large B cell lymphoma (D...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N7/01C12N15/867C12N5/10A61K39/00A61K48/00A61P31/00A61P35/00A61P9/00
CPCA61K48/005A61P9/00A61P31/00A61P35/00A61K39/001129C07K14/35C07K16/2809C07K16/32C07K2317/622C07K2319/02C07K2319/03C07K2319/21C12N7/00C12N15/86C12N2740/15021C12N2740/15043
Inventor 不公告发明人
Owner FUJIAN MEDICAL UNIV
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