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PIWI protein induced by nucleotide fragment and provided with specific endonuclease activity

A nuclease and fragment technology, applied in the field of PIWI protein, can solve problems such as the lack of endonuclease activity

Active Publication Date: 2019-05-10
THE FIRST INST OF OCEANOGRAPHY SOA +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In some Argonaute proteins, the cleavage-active amino acid in PIWI is mutated, resulting in loss of Argonaute protein endonuclease activity

Method used

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  • PIWI protein induced by nucleotide fragment and provided with specific endonuclease activity
  • PIWI protein induced by nucleotide fragment and provided with specific endonuclease activity
  • PIWI protein induced by nucleotide fragment and provided with specific endonuclease activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0040] Example 2: Cutting of PIWI Proteins to Related Plasmids

[0041] 1. PET28PIWI plasmid extraction

[0042] Plasmid extraction was carried out using Sangon Bioengineering Co., Ltd. Column Plasmid DNA Mini-Extraction Kit to extract the plasmids of PET28PIWI and pUC57. The two plasmids have a homologous region of about 800bp. The extracted plasmids were analyzed for quality and quantified by agarose gel electrophoresis.

[0043] 2. Cutting of related plasmids by PIWI nuclease

[0044] After the relevant plasmid is transferred into the host cell, a large number of small DNA / RNA fragments complementary to the plasmid itself will be produced, and the expressed PIWI protein can bind to these small DNA / RNA fragments and produce a shearing effect on the plasmid itself. The PIWI protein was mixed with the plasmid (the plasmid expressing the protein PET28-PIWI) at a molar ratio of 10:1, and reacted at 37°C for 4 hours, and the shearing efficiency was analyzed. When the molar rat...

Embodiment 3

[0045] Example 3: Analysis of PIWI-bound short nucleotide fragments and the guiding effect of 5' phosphorylated DNA fragments on PIWI nuclease directional cleavage

[0046] 1. Type analysis of short nucleotide fragments combined with PIWI

[0047] Treat PIWI with proteinase K, and degrade PIWI completely at 55°C for 2 hours; extract the above degradation products with phenol chloroform, and purify the small nucleotide fragments combined with PIWI; use RNase or DNase to treat the small nucleotide fragments respectively Processing was performed to determine whether PIWI bound DNA or RNA. The results showed that no small nucleotide fragments appeared in the electrophoresis of samples added with RNase, but small nucleotide fragments appeared in the electrophoresis of samples added with DNase, indicating that PIWI binds small RNAs in vivo ( Figure 4 ).

[0048] 2. Design of 5' phosphorylated DNA fragments

[0049] According to the sequence of the pEGFP plasmid, design a 5' phos...

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Abstract

The invention provides a nuclease induced by phosphorylated small DNA and provided with endonuclease activity. The nuclease with a PIWI structure domain particularly has the function of cutting RNA. Although PIWI coming from thermophilic bacteria can cut DNA double strands, the PIWI can only exert the effect at the temperature higher than 65 DEG C. The found PIWI can cut the DNA double strands atthe temperature of 37 DEG C under a mild condition, and accordingly the genetic engineering can be operated more conveniently.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a PIWI protein with endonuclease activity guided by phosphorylated nucleotide fragments (DNA / RNA). Background technique [0002] The PIWI domain is a component of Argonaute proteins and is usually located at the C-terminus of Argonaute proteins. The PIWI family contains a conserved structure consisting of a central PAZ domain and a C-terminal PIWI domain. The PIWI domain has an RNaseH-like folded structure, including a catalytically active tetramer Asp-Glu-Asp-X (X represents Asp, or Asn, or His). The role of Ago protein and nucleotides mainly depends on the binding of amino acid residues on PIWI to the phosphodiester base of DNA / RNA that acts as a guide to cut the target strand. In some Argonaute proteins, the cleavage-active amino acid in PIWI is mutated, resulting in loss of Argonaute endonuclease activity. [0003] The reported PIWI domain basically...

Claims

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Application Information

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IPC IPC(8): C12N9/16C12N15/70
Inventor 郑明刚王玲郑立王闻曲凌云郑择芃马粒雅
Owner THE FIRST INST OF OCEANOGRAPHY SOA
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