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schistosoma japonicum miRNA and application thereof

A schistosomiasis, selected technology, applied in the field of biomedicine and parasites, can solve the problem that researchers have little understanding of miRNA function

Inactive Publication Date: 2011-04-20
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, some miRNAs have been found in Schistosoma japonicum, the pathogen of schistosomiasis, which is a serious threat to human health. few

Method used

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  • schistosoma japonicum miRNA and application thereof
  • schistosoma japonicum miRNA and application thereof
  • schistosoma japonicum miRNA and application thereof

Examples

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preparation example Construction

[0092] The preparation of the miRNA chip can adopt conventional manufacturing methods of biochips known in the art. For example, if the solid-phase carrier is a modified glass slide or silicon wafer, and the 5′ end of the probe contains an amino-modified poly-dT string, the oligonucleotide probe can be prepared into a solution, and then it can be spotted with a spotter The miRNA chip of the present invention can be obtained by arranging the modified glass slide or silicon wafer into a predetermined sequence or array, and then placing it overnight for fixation. If the nucleic acid does not contain amino modification, the preparation method can also refer to: "Gene Diagnosis Technology-Non-Radioactive Operation Manual" edited by Wang Shenwu; JLerisi, VRIyer, POBROWN. Exploring themetabolic and genetic control of gene expression on a Genomic scale. Science, 1997; 278: 680 and Ma Liren, edited by Jiang Zhonghua. Biochip. Beijing: Chemical Industry Press, 2000, 1-130.

[0093] The ma...

Embodiment 1

[0096] Example 1. Obtaining miRNA

[0097] 1. Extraction and purification of small RNA

[0098] The cercariae of Schistosoma japonicum were infected with experimental animals (New Zealand white rabbits or Kunming rats) through the patch, and the animals were dissected 21 days later to obtain young worms.

[0099] The schistosoma juvenile worm specimen was ground into powder with liquid nitrogen, and the total RNA was extracted with Trizol (Invitrogen). Adjust the concentration of non-degraded total RNA to 1mg / ml with DEPC treated water, add 0.5M NaCl and 5% PEG8000, mix well, precipitate at room temperature for 10min, 12000rpm, 4℃, 15min, take the supernatant, add 2.5 Double the volume of absolute ethanol, mix well, and precipitate at -20°C for more than 2 hours. Then 13000rpm, 4℃, 30min. The supernatant was discarded, and the precipitate was dried at room temperature for 5 min. Add appropriate amount of DEPC-treated water or formamide to dissolve RNA, determine the concentration...

Embodiment 2

[0120] Example 2. The effect of miRNA antisense oligonucleotides (ASO) on the growth and development of Schistosoma japonicum

[0121] (1) Synthesis of chemically modified miRNA antisense oligonucleotides (ASO)

[0122] The inventors synthesized chemically modified miRNA ASO, transfected with schistosome, and analyzed the growth and development changes of schistosome. The inventors designed a nucleic acid lock (LNA) modified miRNA ASO. The above-mentioned antisense oligonucleotides are completely matched with the corresponding mature miRNAs, and are modified with LNA every 3 bases. Since the chemical structure of LNA and DNA / RNA are completely the same, they can be synthesized on an automatic DNA synthesizer by the phosphoramidate method and obtained by high-performance liquid chromatography (HPLC) purification.

[0123] A total of 33 antisense nucleotides were prepared, of which the antisense oligonucleotide (ASO) sequence shown in SEQ ID NO: n+66 corresponds to the miRNA shown in...

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Abstract

The invention relates to schistosoma japonicum micro-ribonucleic acid (miRNA) and application thereof. Particularly, the invention discloses new miRNA which is separated out from schistosoma japonicum, wherein the miRNA can be specifically combined with a corresponding part of messenger RNA (mRNA) to influence the transcription effect of the corresponding mRNA so as to influence the expression of a corresponding gene. The invention also provides application of the identified schistosoma japonicum miRNA, a precursor sequence thereof, antisense oligonucleotide for adjusting the expression of the miRNA and the like in diagnosis, prevention and treatment of the schistosoma japonicum.

Description

Technical field [0001] The present invention belongs to the field of biomedicine and parasites; more specifically, the present invention relates to a class of miRNAs isolated from Schistosoma japonicum, their precursors and antisense oligonucleotides, and their use in preparing compositions for inhibiting the growth of Schistosoma japonicum use. Background technique [0002] MicroRNA (miRNA) is a class of highly conserved endogenous small regulatory RNAs in eukaryotes, with a length of about 18 to 26 nucleotides. It can cause target mRNA degradation or translation inhibition by specific base pairing with target mRNA, and regulate gene transcription. After expression. It is derived from the initial transcription product of long-chain RNA (Pri-miRNA) with a length of about 1000bp. The Pri-miRNA molecule is cleaved by Drosha enzyme in the nucleus to form a miRNA precursor with a stem-loop structure of about 60-80 nt (Pre-miRNA). miRNA). After the pre-miRNA is transported to the c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11A61K48/00A61P33/12
Inventor 潘卫庆汪章勋薛向阳孙军
Owner TONGJI UNIV
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