Japanese blood fluke miRNAs and applications thereof

A technology for schistosomiasis, sequence, applied in the fields of biomedicine and parasites

Active Publication Date: 2010-06-16
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, although a large number of miRNAs have been discovered, the pathogen of schistosomiasis, which seriously endangers human health, is still blank.

Method used

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  • Japanese blood fluke miRNAs and applications thereof
  • Japanese blood fluke miRNAs and applications thereof
  • Japanese blood fluke miRNAs and applications thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0085] 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 amino-modified poly dT strings, the oligonucleotide probe can be formulated into a solution, and then spotted with a spotting instrument. The miRNA chip of the present invention can be obtained by arranging in a predetermined sequence or array on a modified glass slide or a silicon chip, and then fixing it by standing overnight. If the nucleic acid does not contain amino modification, its preparation method can also refer to: "Gene Diagnosis Technology-Non-radioactive Operation Manual" edited by Wang Shenwu; J.L.erisi, V.R.Iyer, P.O.BROWN. Exploring the metabolic 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.

[0...

Embodiment 1

[0090] Example 1. Isolation and purification of Schistosoma japonicum small RNA

[0091] The cercariae of Schistosoma japonicum were infected with experimental animals (New Zealand white rabbits or Kunming rats) by patches, and the animals were dissected after 42 days to obtain adults.

[0092] Adult samples of Schistosoma japonicum were ground into powder with liquid nitrogen, and total RNA was extracted with Trizol (Invitrogen). Treat the water with DEPC to adjust the concentration of non-degraded total RNA to 1mg / ml, add NaCl and 5% PEG8000 with a final concentration of 0.5M, mix well, precipitate at room temperature for 10min, 12000rpm, 4°C, 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°C, 30min. The supernatant was discarded, and the precipitate was dried at room temperature for 5 min. Add an appropriate amount of DEPC-treated water or formamide to dissolve RNA, mea...

Embodiment 2

[0093] Example 2. Construction of a cDNA library for small RNAs

[0094] Use 15% urea denatured PAGE gel to recover small RNAs of 18-26 nt, respectively use T4 RNA ligase (MBI) to add 3'-linker 5'-p acuGTAGGCACCATCAA (SEQ ID NO: 16) p-3' and 5'-linker 5'-ATCGTaggcaccugaaa-3' (SEQ ID NO: 17) (wherein, lowercase is RNA, uppercase is DNA, p refers to phosphate group), then use RT primer (5'-ATTGATGGTGCCTAC-3' (SEQ IDNO: 18) ) to reverse transcribe the adapter-added small RNA to obtain cDNA. PCR primers (5' primer: 5'-ATCGTAGGCACCTGAAA-3' (SEQ ID NO: 19), 3' primer: 5'-ATTGATGGTGCCTACAG-3' (SEQ ID NO: 20)) were designed according to the RT primer and the RNA linker. The obtained cDNA was used as a template for PCR amplification, and the PCR product was digested with BshN I (MBI), ligated to itself, filled in again, connected to the T vector pMD18-T plasmid (Takara), screened positive clones by PCR, and sequenced.

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Abstract

The invention relates to Japanese blood fluke miRNAs. In the invention, a new type of miRNAs is firstly separated from blood flukes, and the expression of the miRNAs has the characteristic of specificity in the life history of blood flukes. Antisense oligonucleotides of MiRNAs modified by cercaria transfection in the infective stage of blood flukes can affect the subsequent growth of blood flukes. The invention provides approved Japanese blood fluke miRNAs and a precursor sequence thereof, and applications of antisense oligonucleotides and the like for regulating the expression of miRNAs in prevention and control of blood flukes.

Description

technical field [0001] The invention belongs to the fields of biomedicine and parasites; more specifically, the invention relates to miRNA isolated from schistosomiasis, its precursor and antisense oligonucleotide, and their use in preparing a composition for inhibiting schistosomiasis. Background technique [0002] MicroRNA (MiRNA) is a kind of highly conserved endogenous small regulatory RNA in eukaryotes, with a length of about 18-26 nucleotides, which causes target mRNA degradation or translation inhibition by specific base pairing with target mRNA. Regulates post-transcriptional expression of genes. It is derived from the long-chain RNA initial transcription product (Pri-miRNA) with a length of about 1000 bp. Pri-miRNA molecules are cleaved by Drosha enzyme in the nucleus to form miRNA precursors (Pre-miRNA) with a stem-loop structure of about 60-80 nt in length. miRNA). After the pre-miRNA is transported to the cytoplasm, it is further cut into a double-stranded miRN...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/11A61K31/7088A61P33/12
CPCY02A50/30
Inventor 潘卫庆薛向阳孙军汪章勋
Owner TONGJI UNIV
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