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Transduction peptide-dsRNA (double-stranded ribonucleic acid) binding domain fusion protein and application thereof

A fusion protein and binding domain technology, applied in the field of transduction peptide-dsRNA binding domain fusion proteins, can solve problems such as restricting application, weakening penetration function, etc., to achieve the effect of efficient binding

Inactive Publication Date: 2014-04-23
李华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the study, it was found that negatively charged siRNA easily combined with positively charged transduction peptide, resulting in further aggregation and precipitation of siRNA / transduction peptide polymer (Meade & Dowdy, 2007), or negatively charged siRNA neutralized the positive transduction peptide. Electrically, weaken or terminate the cell membrane penetration function of the transducing peptide (Meade & Dowdy, 2008; Veldhoen, Laufer, Trampe, & Restle, 2006), thus limiting its application in siRNA transduction

Method used

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  • Transduction peptide-dsRNA (double-stranded ribonucleic acid) binding domain fusion protein and application thereof
  • Transduction peptide-dsRNA (double-stranded ribonucleic acid) binding domain fusion protein and application thereof
  • Transduction peptide-dsRNA (double-stranded ribonucleic acid) binding domain fusion protein and application thereof

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Embodiment 1

[0023] Construction of Hph1-Hph1-dsRBD Gene

[0024] Synthesize TAT-dsRBD and Hph1-Hph1-TAT-TAT-dsRBD genes, and add NheI, PstI and EcoR I restriction sites to them, see the gene structure diagram ( figure 1 ). First clone Hph1-Hph1-TAT-TAT-dsRBD into PET28b vector through NheI and EcoR I restriction sites, then cut TAT-dsRBD and PET28b-Hph1-Hph1-TAT-TAT-dsRBD genes through PstI and EcoR I, The obtained dsRBD and PET28b / Hph1-Hp were further ligated under the action of ligase to produce PET28b / Hph1-Hph1-dsRBD expression vector. The resulting expression vector was identified by Nhe I and EcoRI digestion figure 2 , and verified by enzyme digestion that the correct expression vector was sequenced by Eurofin Company in Germany. The sequencing primers were T7TAA TAC GAC TCA CTA TAG GG primers. The sequencing result was the Hph1-Hph1-dsRBD gene sequence. See the sequence table for the sequence.

[0025] TAT-dsRBD

[0026] CGCGGATCCCGCAAGAAACGCCGCCAGCGCCGCCGCAGATCTCTGCAGCCATGGTTCTT...

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Abstract

The invention belongs to the field of gene engineering and in particular relates to a transduction peptide-dsRNA (double-stranded ribonucleic acid) binding domain fusion protein and an application thereof. The fusion protein is formed by two cell membrane penetrating peptides Hph1 and a dsRNA binding domain in dsRNA-dependent protein kinase (PKR) and has molecular weight of 18kD. The nucleotide sequence of the fusion protein is shown in SEQ ID NO.1, and the protein sequence of the fusion protein is shown in SEQ ID NO.2. The siRNA (small interfering RNA) new vector with an efficient siRNA transduction function is constructed by combining the transduction function of the transduction peptides with the high siRNA affinity of the dsRNA binding domain for the first time. The siRNA new vector has better effects than the vectors on the market at present, and provides extensive prospects for treatment of siRNA-mediated diseases.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a transduction peptide-dsRNA binding domain fusion protein and application thereof. Background technique [0002] Cell transduction peptides are short peptides composed of 4-30 arginine- or lysine-rich peptides, which have the ability to penetrate cell membranes (Gautam et al., 2012; Lindgren, Llbrink, M., A, & Langel , 2000), in 1988 the transduction peptide Tat (trans activity transcript) was first discovered in HIV virus (Frankel & Pabo, 1988). Cell transduction peptides can transfect biological macromolecules (molecular weight greater than 180kDa) (Funhoff et al., 2004), peptides, plasmids, nanoparticles, etc. to penetrate the cell membrane and the blood-brain barrier (Lindgren, Gallet, et al., 2000) Cell membranes that are difficult for ordinary carriers to penetrate. In the past 20 years of research, transduction peptides have been extensively studied and applied, but t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/87
Inventor 李华徐东宇
Owner 李华