Unlock instant, AI-driven research and patent intelligence for your innovation.

Small disturbance RNA molecule expression carrier for differentially inhibiting GFAP protein expression and its construction method and use thereof

An expression vector and protein expression technology, applied in the direction of recombinant DNA technology, the use of vectors to introduce foreign genetic material, and pharmaceutical formulations, can solve the problems of high experimental cost, many research interference links, and low inhibition efficiency

Inactive Publication Date: 2012-07-11
THE SECOND PEOPLES HOSPITAL OF SHENZHEN
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the past, scholars at home and abroad have applied antisense nucleic acid technology to the inhibition of glial scars. However, due to the high cost of experiments, long experiment cycle, many research interference links, and low inhibition efficiency, traditional antisense RNA technology and ribozymes are natural. Unfavorable factors, so people hope that the emergence of new antisense nucleic acid technology

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Small disturbance RNA molecule expression carrier for differentially inhibiting GFAP protein expression and its construction method and use thereof
  • Small disturbance RNA molecule expression carrier for differentially inhibiting GFAP protein expression and its construction method and use thereof
  • Small disturbance RNA molecule expression carrier for differentially inhibiting GFAP protein expression and its construction method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: GFAP-specific shRNA transcription template sequence design and synthesis

[0023] Log on to Genebank, retrieve the full coding sequence of GFAP gene mRNA, the gene sequence number is GI: 8393430, use Ambion's (www.ambion.com) siRNA Target Finder tool, and refer to the siRNA design principles, start codon from the open reading frame of the target gene After 75 to 100 bases downstream of "ATG" or before 100 bp before the terminator, search for the 19 bases after the "AA" duplex sequence as a potential siRNA target site. When designing siRNA, the 5' and 3' non-coding regions of GFAP gene mRNA were not targeted, and the obtained sequences were analyzed, and the GC content was selected between 40% and 55%, and searched by BLAST software in Genebank to exclude homology with other coding genes sequence; and introduced BamH I, HindIII restriction site, 9bp stem-loop structure and transcription termination sequence to form a short hairpin shRNA molecule, designed and ...

Embodiment 2

[0027] Example 2: Construction and identification of eukaryotic expression plasmids for GFAP-specific siRNA molecules

[0028] In the pGFAP shRNA fragment design, the restriction sites of EcoR1, SacI and SalI (purchased from Promega Company) were inserted in the pGFAP-1, 2, and 3 templates respectively, and inserted into the plasmid pGenesil (purchased from Wuhan Jingsai Bioengineering Technology Co., Ltd. company, carrying the red fluorescent protein expression system) between HindIII and BamHI of the multiple cloning site. After BamH1, there are EcoRI and Sacl enzyme cutting sites. If the directional cloning is correct, pGFAP-1 and 2 can be digested by EcoRI and SalI respectively to produce a small DNA band of about 400bp; while pGFAP-3 can only be linearized by SalI (results in Figure 1); identified by enzyme digestion Analysis showed that the recombinant plasmids pGFAP-1, 2, 3 and the negative control HK all met the design requirements. The four groups of recombinant pla...

Embodiment 3

[0029] Example 3: Primary culture and transfection of spinal cord-derived astrocytes

[0030] Take out 2-3d Wister rats after birth, take the spinal cord tissue of suckling rats under aseptic conditions, peel off the meninges and blood vessels under a dissecting microscope, digest with 0.125% trypsin, and use the differential adhesion method twice to remove the contamination of fibroblasts , with DMEM / F12 medium containing 20% ​​fetal bovine serum, cultured at 37°C and 5% CO2 for 8--9 days, after removing the contamination of oligodendrocytes by shaking method, the purity of the cells was confirmed to be 95% by immunohistochemistry %, the cells can be transfected. The day before transfection, press 1×10 6 / ml into a six-well plate (the wells are pre-coated with polylysine slides), incubated for 24 hours, and when the cell fusion reached 90%, refer to the Invitrogen product instructions, and take 4ug pGFAP-shRNA and Control-shRNA plasmids respectively Mix with 8-12ul Lipofect...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a small interfering RNA molecule expression vector and a construction method and use thereof expressed by a specifically inhibited GFAP protein. The target of the invention is to provide a small interfering RNA molecule expression vector pressed by a specifically inhibited GFAP protein and the applications thereof for clinical treatment drug. The small interfering RNA is a double-chain RNA sequence with a sense strain of SEQ ID No.1 in the sequence list and an anti-sense strain of SEQ ID No.2 of the sequence list, or other nucleotide sequence originates from GFAP gene code region and designed according the statement of the instruction book. The constructed small molecular interfering RNA expression vector can be used for the treatment of relevant symptoms caused by abnormal GFAP gene expression. The invention has comparatively greater application value and prospect in medicine and bio-pharmacy fields.

Description

technical field [0001] The present invention relates to a method and application for inhibiting the expression of GFAP protein by using small interfering RNA molecules, especially a method for expressing small interfering RNAs targeting GFAP gene with eukaryotic expression vectors such as adenovirus and the use of the small interfering RNA molecules expressed by this method For the treatment of GFAP protein-related symptoms. Background technique [0002] The repair of central nervous system injury has always been a difficult point in the field of neuroscience, and the factors that hinder local nerve regeneration in the damaged area are extremely complex. glial scar, which constitutes a space barrier that hinders the extension of axons and the effective diffusion of nutrient factors, which is unfavorable to the repair of the central nervous system; as the internal environment of the injury gradually stabilizes, astrocytes express a large number of proteoglycan molecules that ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66C07H21/02C12N15/85A61K48/00A61P25/00
Inventor 高明勇肖建德李振宇闫洪印
Owner THE SECOND PEOPLES HOSPITAL OF SHENZHEN