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Slow virus vector, preparation thereof and application thereof

A technology of lentiviral vector and short hairpin, which is applied in the field of lentiviral vector, can solve the problem of reducing expression and achieve the effect of inhibiting growth

Inactive Publication Date: 2009-11-11
SUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Moreover, there is no direct evidence that reducing the expression of the fusion gene in 293T cells will certainly reduce the expression of the FoxP3 gene in natural regulatory T cells

Method used

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  • Slow virus vector, preparation thereof and application thereof
  • Slow virus vector, preparation thereof and application thereof
  • Slow virus vector, preparation thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] According to the mouse FoxP3 mRNA sequence (NCBI NM_054039), use BLOCK-iT RNAiDesigner online software to design a 50nt nucleotide sequence as the RNAi target. The sequence number is: ShRNA-FoxP3.

[0028] Sense strand: 5’-CAC CGC CAT GGC AAT AGT TCC TTC CCG AAG GAAGGA ACT ATT GCC ATG GC-3’

[0029] Antisense strand: 5’-AAA AGC CAT GGC AAT AGT TCC TTC CTT CGG GAAGGA ACT ATT GCC ATG GC-3’

Embodiment 2

[0030] Embodiment two: see Figure 1 ~ Figure 4 , refer to the instructions of the Lentivirus-shRNA system to construct a recombinant plasmid

[0031] Take 10 μg of shRNA fragments synthesized and purified in Example 1, and anneal to form complementary double strands. The pENTR / U6 plasmid and shRNA were connected with T4 ligase at 1:2, transformed into the competent strain One ShotTOP10, and the site was screened with kanamycin to form pENTR / U6-FoxP3-ShRNA (such as figure 1 shown). Using ABI-7700 sequencing, it was confirmed that the synthetic sequence was correct and there was no gene mutation after comparison with the synthetic fragment.

[0032] The pENTR / U6-FoxP3-ShRNA with the correct sequence was recombined with pLenti6 / BLOCK-iT-DEST using BLOCK-iT LentiviralRNAi Expression System (such as figure 2 shown), the recombinant plasmid pLenti-FoxP3-shRNA (such as image 3 shown), the whole process is as follows Figure 4 shown.

Embodiment 3

[0033] Example 3: Prepare recombinant lentivirus pLenti-FoxP3-shRNA according to the instructions of the Lentivirus-shRNA system

[0034] (1) Inoculate 10 6 293FT cells were subcultured at 1:3 when the cells grew to 70%-80% confluence, and DMEM (GIBCO) containing 10% calf serum was used as the culture medium at 37°C in 5% CO 2 Cultivate in the incubator for 24h.

[0035] (2) When the cells reach 70% confluence, the pLenti-FoxP3-shRNA plasmid and the packaging plasmid of the lentiviral vector are transfected with calcium phosphate co-precipitation.

[0036] (3) Change the cell culture medium after the cells grow for 24 hours, and place at 37°C with 5% CO 2 Cultivate in an incubator, obtain the virus supernatant after 24 hours, and replace the cell culture medium.

[0037] (4) Harvest the virus supernatant 72 hours after transformation, and treat the cell culture dish with bleach to destroy the virus producing cells.

[0038] (5) Ultracentrifuge the virus supernatant at 50,000...

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Abstract

The invention discloses a slow virus vector. Preparation and application of the slow virus vector comprise the following steps: firstly, aiming at a FoxP3 gene sequence of a mouse, designing specific short hairpin RNA which comprises a sense strand and an antisense strand, and then carrying the short hairpin RNA into pENTR / U6 plasmids; then using a Lenti virus shRNA expression vectors system to carry out slow virus package in 293 FT cells to obtain the slow virus vector carried with the short hairpin RNA; and introducing the slow virus vector carried with the short hairpin RNA into Treg cells of CD4+CD25+ so as to reduce the function of the Treg cells and inhibit the growth of melanoma.

Description

technical field [0001] The present invention relates to a lentiviral vector, in particular to a lentiviral-mediated RNA interference technology (RNA interference, RNAi) targeting FoxP3 gene, and a lentiviral vector with shRNA. Background technique [0002] FoxP3 is a transcriptional repressor, a member of the forkhead / winged-helix family, and is related to the regulation of cell growth and development. Later, more and more evidence showed that FoxP3 may be a CD4+CD25+Treg immunosuppressive regulator of cell development, Genes that are crucial for differentiation and have very critical roles in the maintenance of their phenotype and function. [0003] Studies have shown that regulatory T cells (Treg) can affect the body's role in tumor monitoring by regulating effector T cells. As the main marker of Treg cells, forkhead / winged helix transcription factor (FoxP3) is necessary for Treg to function and is an important switch for Treg cell development. Studies have found that tr...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/867A61K48/00A61P35/00C12N15/113
Inventor 周翊峰
Owner SUZHOU UNIV
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