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Unique vector coded hair pin structure small interference RNA expression system and its establishing method

A hairpin structure and expression system technology, applied in the biological field, can solve problems such as failure to achieve, difficulty in inserting the encoding target shRNA sequence into the vector, and difficulty in encoding, so as to achieve enhanced effects, improve RNA interference effects, and inhibit HIV replication.

Inactive Publication Date: 2006-09-27
WUHAN CELL MARKER & MACHINE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, modern biotechnology does not meet the requirements of "just enough" biological enzymes
Due to the lack of suitable available enzymes, it is very difficult to insert the target shRNA sequence into the vector
It is more difficult to realize multiple shRNAs encoded in one vector

Method used

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  • Unique vector coded hair pin structure small interference RNA expression system and its establishing method
  • Unique vector coded hair pin structure small interference RNA expression system and its establishing method
  • Unique vector coded hair pin structure small interference RNA expression system and its establishing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The construction method of the single vector expression system encoding multiple hairpin structure small interfering RNAs of the present invention is further described as follows in conjunction with the accompanying drawings

[0057] (1) Digest the pUC18 plasmid with BamHI and EcoRI double enzyme digestion, recover a large piece, and obtain the PUC18 plasmid digested by BamHI and EcoRI double enzyme digestion; (see figure 1 )

[0058] (2) Digest pSilencer with BamHI and EcoRI double enzyme digestion TM 2.1neo Vector, reclaim small fragment, obtain the small fragment that contains U6 promoter, structure is BamHI-U6 promoter-EcoRI; (see figure 2 )

[0059] (3) Insert the small BamHI-U6 promoter-EcoRI structure fragment obtained in the above step (2) into the PUC18 plasmid digested by BamHI and EcoRI double enzyme digestion obtained in the previous step (1), to obtain a HindIII-PaeI-PstI- The recombinant pUC18 plasmid of SalI-XbaI-BamHI-U6 promoter-EcoRI structural u...

Embodiment 2

[0076] Purpose:

[0077] Construct a single vector encoding multiple hairpin structure small interfering RNA (short hairpin RNA, shRNA) expression system; prepare hairpin structure small interfering RNA encoding and blocking two human lymphocyte CD3 receptor genes and two human lymphocyte CD4 receptor genes , detect the expression of CD3 and CD4 on the cell membrane after being introduced into the cells, and compare the effect difference between a single vector encoding multiple hairpin structure small interfering RNAs and a single vector encoding one hairpin structure small interfering RNA.

[0078] Experimental Materials:

[0079] Product names of reagents and laboratory tools

The company or unit that provides the product

[0080] PUC18 plasmid

pSilencer TM 2.1neo Vector

molta4 cells

EcoRI

BamHI

Hind III

pstI

wxya

SalI

PaI

T4DNA Ligase

T4 Polynucleotide Kinase

Mung bean enzyme

DH...

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Abstract

This invention realizes that one bearer encodes multiple hair-pin interferences RNA at the same time. That one bearer encodes multiple shRNA can block and interferene different locuses of one destination gene or various destination genes at the same time. It also opens one preparation method that one bearer encodes various dual-chain RNA and establishes one mammalian cell expression vector that encodes multiple dual-chain RNA. This invention develops the technology of RNA interference and improves its effects, and thus has an applicable value to RNA interference technology and short dual-chain RNA as gene medicine.

Description

technical field [0001] The invention relates to biotechnology, in particular to RNA interference technology, in particular to a short interfering RNA expression system and a construction method thereof. Background technique [0002] RNA interference technology was discovered in 1998, and was successfully used in mammalian cells in May 2001, starting the wide application of RNA interference technology. RNA interference technology uses 21nt or 29nt short interfering RNA to block the target gene to achieve the effect of interfering or blocking the target gene. RNA interference is the oldest genetic immune surveillance mechanism in life. Life maintenance and health, anti-virus invasion, anti-tumor occurrence, and maintenance of genetic stability may all be achieved by RNA interference. Therefore, the research on RNA interference and the application of RNA interference technology have attracted unprecedented attention. In the year when RNA interference technology was successful...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/85C12N15/86C12N15/12
Inventor 封江南
Owner WUHAN CELL MARKER & MACHINE TECH