Characterization and application of Novel high-temperature Argonaute protein TpsAgo

A reaction and reaction system technology, applied in the biological field, can solve problems such as the scarcity of circulating tumor DNA, sensitivity, and operating cost limitations

Active Publication Date: 2021-08-03
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although circulating tumor DNA is a good tumor tissue substitute sample, due to the scarcity of circulating tumor DNA, the detection of circulating tumor DNA requires extremely sensitive techniques
The current detection technology mainly relies on next-generation sequencing and digital PCR technology, but they all have certain limitations in terms of sensitivity and operating cost

Method used

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  • Characterization and application of Novel high-temperature Argonaute protein TpsAgo
  • Characterization and application of Novel high-temperature Argonaute protein TpsAgo
  • Characterization and application of Novel high-temperature Argonaute protein TpsAgo

Examples

Experimental program
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Effect test

Embodiment 1

[0226] Embodiment 1: Obtaining of TpsAgo gene sequence

[0227] In the database, the amino acid sequence of known PfAgo was searched for similarity, some amino acid sequences with high sequence consistency were selected, analyzed by MEGA software, a homologous evolution tree was constructed, and TpsAgo was selected as a candidate enzyme. The amino acid sequence of TpsAgo (WP_060384876.1) and the corresponding gene sequence (NZ_CP014142.1) encoding the protein were obtained. After the gene sequence was synthesized by codon optimization, it was cloned into pET28a expression vector.

Embodiment 2

[0228] Example 2: Heterologous expression and purification of TpsAgo protein

[0229] The above TpsAgo-pET28a prokaryotic expression plasmid was introduced into E.coli BL21(DE3) to obtain the TpsAgo-pET28a / E.coli BL21(DE3) prokaryotic expression strain. The expression strain E.coliBL21(DE3) containing the recombinant plasmid TpsAgo-pET28a was inoculated in LB medium containing 50 μg / mL kanamycin, and cultivated to OD at 37°C and 220rpm on a shaker 600 Between 0.6-0.8, add IPTG with a final concentration of 0.4-0.6mM, continue to culture at 18°C, 200rpm shaker for 16-20h, and induce the expression of TpsAgo protein. Collect the cells by centrifugation, resuspend the cells in a resuspension buffer (containing 20mM Tris-HCl, pH around 8.0, 1M NaCl), then crush the cells by high pressure, and centrifuge to obtain the supernatant. Using Ni-NTA column to affinity purify the protein, the eluate is concentrated by ultrafiltration, desalted and other steps to obtain the purified prote...

Embodiment 3

[0231] Example 3: Alignment of TpsAgo with other known Ago sequences

[0232] In this example, multiple sequence alignments were performed between TpsAgo and partially characterized Ago.

[0233] The results showed that TpsAgo had the least number of amino acids and the smallest protein molecular weight. According to literature reports, the targeted cleavage of all catalytically active Ago proteins is mediated by a conserved DEDX (X stands for histidine, aspartic acid or asparagine) quadruple. Through sequence comparison, it can be found that there is a DEDD quadruple in TpsAgo ( figure 1 ). Therefore, it is further speculated that it may have nuclease catalytic activity, which requires further identification and characterization in vitro.

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Abstract

The invention provides characterization and application of a novel high-temperature Argonaute protein. Specifically, the invention provides a nucleic acid cleavage system, which is characterized in that the nucleic acid cleavage system comprises: (a) guide DNA (gDNA); (b) a programmable endonuclease Argonaute (Ago); and (c) optionally a reporter nucleic acid, wherein if the reporter nucleic acid is cleaved, the cleavage can be detected. In addition, the invention also provides a system and a method for enriching and detecting low-abundance target nucleic acid based on the programmable endonuclease TpsAgo provided by the invention. The detection system and method provided by the invention can specifically and effectively enrich and efficiently detect low-abundance nucleic acid, and the programmable endonuclease TpsAgo provided by the invention has strong gene manipulation potential.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the characterization and application of a novel high-temperature Argonaute protein TpsAgo. Background technique [0002] The Argonaute (Ago) protein was first mentioned in a study describing a mutant of Arabidopsis thaliana. Ago proteins are key players in the eukaryotic RNA interference (RNAi) pathway that regulate gene expression post-transcriptionally, thereby defending against host-invading RNA viruses and preserving genome integrity. The reported Ago proteins are mainly divided into two types: eukaryotic Ago (eAgo) and prokaryotic Ago (pAgo). [0003] Prokaryotic Ago can bind to single-stranded guide DNA or RNA, and catalyze the cleavage of target DNA or RNA that is complementary to guide. Different from the CRISPR / Cas system, prokaryotic Ago does not need a PAM sequence when cutting the target nucleic acid chain. It can bind to the guide DNA or RNA complementary t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816C12Q1/6806C12N9/22C12N15/55
CPCC12Q1/6816C12Q1/6806C12N9/22C12Q2521/327C12Q2525/161C12Q2565/20C12Q2527/125C12Q2521/513Y02A50/30
Inventor 冯雁孙莹璎郭翔叶星宇李忠磊
Owner SHANGHAI JIAO TONG UNIV
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