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Construction method of RNA interference recombinant lentiviral vector targeting il-33 gene

An RNA interference, recombinant lentivirus technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., to achieve highly specific effects

Inactive Publication Date: 2016-06-29
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no relevant report on lentiviral vector-mediated IL-33 RNA interference technology

Method used

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  • Construction method of RNA interference recombinant lentiviral vector targeting il-33 gene
  • Construction method of RNA interference recombinant lentiviral vector targeting il-33 gene
  • Construction method of RNA interference recombinant lentiviral vector targeting il-33 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0041] (2) mus-IL-33 oligonucleotide sequence 2 in Example 2:

[0042] Sense strand: 5'-gatccGTGCTACTACGCTACTATGTTCAAGAGACATAGTAGCGTAGTAGCACTTTTTTACGCGTg-3',

[0043] Antisense strand: 5'-aattcACGCGTAAAAAAGTGCTACTACGCTACTATGTCTCTTGAACATAGTAGCGTAGTAGCACg-3';

Embodiment 3

[0044] (3) mus-IL-33 oligonucleotide sequence 3 in embodiment 3:

[0045] Sense strand: 5'-gatccGCCATAAGAAAGGAGACTAGTTCAAGACTAGTCTCCCTTTCTTATGGTTTTTTACGCGTg-3',

[0046] Antisense strand: 5'-aattcACGCGTAAAAAACCATAAGAAAGGAGACTAGTCTCTTGAACTAGTCTCCTTTCTTATGGCg-3';

[0047] (4) mus-IL-33 oligonucleotide sequence 4 in embodiment 4:

[0048] Sense strand: 5'-gatccGCCCTGAGTACATACAATGATTCAAGAGATCATTGTATGTACTCAGGGTTTTTTACGCGTg-3',

[0049] Sense strand: 5'-aattcACGCGTAAAAAAACCCTGAGTACATACAATGATCTCTTGAATCATTGTATGTACTCAGGGCg-3'.

[0050] 2. Construction of pLVX-IL-33-shRNA2 recombinant vector

[0051] (1) Lentivirus-shRNA primer annealing

[0052] The double-stranded DNA fragments synthesized in each example were dissolved in TEbuffer (pH 8.0) at a concentration of 100 μM. Take 2 μL each of the corresponding sense strand and antisense strand oligomer solutions, and perform annealing reaction on a PCR machine. The program is as follows: 95°C for 30s; 72°C for 2min; 37°C for 2min; 25...

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Abstract

A double-strand DNA fragment is designed and synthesized aiming at an IL-33 gene and is connected to a lentiviral vector to prepare a recombinant lentivirus plasmid vector. Then the recombinant lentivirus plasmid vector and a virus are packaged into three needed auxiliary vectors and cotransfected into a 293T cell to obtain a target RNA-interfered recombinant lentivirus vector for the IL-33 gene, thus laying good foundations for further study of the IL-33. The lentiviral vector has low immunogenicity and is capable of infecting cells in the division phase and the non-division phase, integrating fragments carried itself into a host cell genome, stably expressing siRNA in various cells of mammal and inhibiting gene expression for long term. Compared with a plasmid vector and other virus vectors, the RNA interference mediated by a lentivirus is characterized by being efficient, stable, and strong in specificity. The construction method provides a novel thought for in-vivo gene therapy and study of functions of the IL-33 gene.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for constructing a recombinant lentiviral vector targeting IL-33 gene RNA interference. Background technique [0002] Interleukin-33, or interleukin-33, or IL-33 for short, is the eleventh member of the IL-1 family discovered by Schmitz et al. through computer-aided means, also known as IL-1F11. The human IL-33 gene is located on chromosome 9p24.1, and the corresponding mouse gene is located on chromosome 19qC1. In humans and mice, the IL-33 gene encodes polypeptides consisting of 270 and 266 amino acid residues, respectively, and the corresponding full-length protein molecular weights are 30kD and 29.9kD, respectively. IL-33 is widely distributed in various organ systems of the body, and is mainly expressed in non-hematopoietic cells, including fibroblasts, adipocytes, smooth muscle cells, endothelial cells, bronchial and intestinal epithelial cells, et...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867
Inventor 李燕高巧艳李明才高雪明袁仙丽彭笑
Owner NINGBO UNIV