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SgRNA and plasmid group of targeted IL-16 gene, gene knockout method and application thereof

A technology of plasmid and gene, applied in the field of genetic engineering

Active Publication Date: 2021-06-15
THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the impact of stably knocking out the IL-16 gene in leukemia tumor cells in the prior art

Method used

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  • SgRNA and plasmid group of targeted IL-16 gene, gene knockout method and application thereof
  • SgRNA and plasmid group of targeted IL-16 gene, gene knockout method and application thereof
  • SgRNA and plasmid group of targeted IL-16 gene, gene knockout method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] A plasmid set for knocking out the IL-16 gene, the construction method is as follows:

[0052] (1) After px330 was digested with BbsⅠ, the main large fragment was recovered from the gel for later use.

[0053] (2) The designed sgRNA is as follows:

[0054] sgRNA1-S: CACC GCAGCTGGCAGACACATCGG (SEQ ID No. 1);

[0055] sgRNA1-A: AAAC CCGATGTGTCTGCCAGCTGC (SEQ ID No. 2);

[0056] The sgRNA1-S and sgRNA1-A were annealed separately to form a double-stranded structure containing BbsI cohesive ends. 10 μl system, containing 1 μl annealed PBbuffer, 1 μl each of sgRNA1-S and sgRNA1-A (the concentration is 100 μmol L -1 ), annealed at 95°C for 5 minutes, and set aside.

[0057] (3) Ligation: 10 μl system, containing 50 ng of vector, 10:1 molar ratio of insert fragment to vector, 1 μl of T4Buffer, 1 μl of T4 ligase, overnight at 16°C.

[0058] (4) Transform the ligation product, pick clones for sequencing identification, and obtain sgRNA1-S plasmid and sgRNA1-A plasmid.

...

Embodiment 2

[0098] Using the same method as in Example 1 to construct a plasmid set, the only difference is that sgRNA is as follows:

[0099] sgRNA2-S: CACC GCTGCTCAACTCCAAGCAGC (SEQ ID No. 3);

[0100] sgRNA2-A: AAAC GCTGCTTGGAGTTGAGCAGC (SEQ ID No. 4).

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Abstract

The invention relates to the technical field of gene engineering, in particular to a sgRNA and a plasmid group of targeted IL-16 gene, a gene knockout method and application thereof The sgRNA disclosed by the invention comprises sgRNA1 or sgRNA2; the sgRNA 1 comprises sgRNA 1-S and sgRNA 1-A; the sgRNA 2 comprises sgRNA 2-S and sgRNA 2-A. The sgRNA of targeted IL-16 gene provided by the invention not only can stably knock out the IL-16 gene in leukemia tumor cells, but also can be used for preparing drugs for inhibiting tumor proliferation.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a sgRNA targeting IL-16 gene, a plasmid group and a gene knockout method and application. Background technique [0002] IL-16 is also known as a lymphocyte chemokine. At present, no other cytokines in the same family have been found, and it is highly conserved among different species. The human IL-16 gene has a full length of 153kbp and is located on the human chromosome 15q26.3. IL-16 precursor protein contains 631 amino acids with a molecular weight of 80kDa. Once activated, the precursor IL-16 stored in the cell will be cleaved by caspase-3 molecules, and the part containing the N-terminal domain forms a non-secreted protein with a molecular weight of 60kDa; the part containing the C-terminal domain forms a molecular weight of 60kDa 20kDa secreted protein. In lymphocytes, molecules containing N-terminal domains can bind to transcription factors, thereby affecti...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N5/10A61K31/7105A61P35/00A61P35/02
CPCC12N15/1136C12N15/85A61K31/7105A61P35/00A61P35/02C12N2310/20
Inventor 王一郭梅蔡博莫丹胡锴勋黄雅静刘志青刘铁强黄珊李冰霞
Owner THE FIFTH MEDICAL CENT OF CHINESE PLA GENERAL HOSPITAL
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