MicroRNA inhibitor, microRNA inhibitor expression vector, building method of microRNA inhibitor expression vector and application of microRNA inhibitor expression vector

A technology for expression vectors and construction methods, applied in the field of biomedicine, can solve the problems of low efficiency, large side effects, high cost, etc., and achieve the effects of high vector utilization, shortened construction cycle, low usage and toxicity

Inactive Publication Date: 2015-12-09
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a microRNA inhibitor, a microRNA inhibitor expression vector and its construction method and application. The microRNA inhibitor is a miRNA adhesion molecule (double miRNAadhesionnucleotide, dMAN), which is based on A class of competitively adsorbed miRNA inhibitors designed based on the principle of miRNA sponge body, which can be used to efficiently adsorb target miRNA, aiming to solve the problems of low efficiency, large side effects or high cost existing in the existing methods of inhibiting miRNA

Method used

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  • MicroRNA inhibitor, microRNA inhibitor expression vector, building method of microRNA inhibitor expression vector and application of microRNA inhibitor expression vector
  • MicroRNA inhibitor, microRNA inhibitor expression vector, building method of microRNA inhibitor expression vector and application of microRNA inhibitor expression vector
  • MicroRNA inhibitor, microRNA inhibitor expression vector, building method of microRNA inhibitor expression vector and application of microRNA inhibitor expression vector

Examples

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Effect test

Embodiment 1

[0068] Example 1 relates to the specific preparation process of pLVX-dMAN.

Embodiment 2

[0069] Example 2 relates to a method for transiently transfecting pLVX-dMAN vectors to inhibit miRNA activity in cells. This method transfects the constructed pLVX-dMAN-miR-195 and pLVX-dMAN-miR-497 into human embryonic kidney via liposomes In epithelial 293A cells, total RNA was collected after 48 hours, the expression levels of miR-195 and miR-497 were detected by qRT-PCR, and the inhibitory effect of dMAN was calculated.

Embodiment 3

[0070] Example 3 relates to the method of packaging pLVX-dMAN into lentivirus-infected cells and inhibiting miRNA activity in cells. In this method, the constructed pLVX-dMAN-miR-322 and pLVX-dMAN-miR-497 were packaged into lentiviral particles, infected with mouse myogenic differentiated C2C12 cells to screen stable transfected cell lines, collected total RNA, and detected miR by qRT-PCR The expression levels of -322 and miR-497 were used to calculate the dMAN inhibitory effect.

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Abstract

The invention discloses a microRNA inhibitor, a microRNA inhibitor expression vector, a building method of the microRNA inhibitor expression vector and application of the microRNA inhibitor expression vector. The microRAN inhibitor is a dMAN (double mRNA adhesion nucleotide), and consists of three parts including a general straight chain complementary sequence, a general stem ring sequence and two miRNA combining region sequences. The built expression vector comprises two dMAN expression frameworks; two identical or different MANs can be transcribed at the same time; the utilization rate of the vector is higher; and the consumption and the toxicity of virus particles are lower. In an aspect of the building method, the dMAN building method is economic; only two pairs of primers of at most 60 basic groups need to be synthesized; and one-step cloning building comprising two MAN structure mRNA inhibition vectors can be completed through twice PCR (Polymerase Chain Reaction) and an enzyme digestion and connecting technology.

Description

technical field [0001] The invention relates to the field of biomedicine, and mainly relates to a microRNA inhibitor, a microRNA inhibitor expression vector, a construction method and application thereof. Background technique [0002] micorRNA (miRNA) is a kind of naturally occurring small non-coding RNA with a length of about 17-25 bases. It widely exists in eukaryotic organisms such as animals, plants, and nematodes, and has a high degree of homology among organisms. The gene encoding miRNA is transcribed into primary miRNA (pri-miRNA) by RNA polymerase in the nucleus, and further becomes mature miRNA (mature miRNA) under the action of enzymes such as Drosha and Dicer. Mature miRNAs form RISC complexes with related proteins such as Ago to regulate the expression of related proteins. The complex is guided by the mature miRNA, and through specific binding to the 3'-UTR of the target mRNA, it degrades the target mRNA or represses its post-transcriptional translation, which u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/66A61K48/00A61P43/00
Inventor 苟德明康康邱惠玲钟嘉胜罗兰
Owner SHENZHEN UNIV
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