Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

miRNA-340 target gene binding sequence, recombinant plasmid and application thereof

A technology that combines sequences and recombinant plasmids, applied in DNA/RNA fragments, genetic engineering, recombinant DNA technology, etc., can solve problems such as intractable diseases and adverse reactions

Active Publication Date: 2021-03-05
SHENZHEN INST OF ADVANCED TECH
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, glucocorticoids and immunosuppressants are commonly used in the treatment of autoimmune diseases. Although they have a certain effect, long-term use will cause serious adverse reactions, and they can only slow down the development of the disease and cannot cure the disease.

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
  • miRNA-340 target gene binding sequence, recombinant plasmid and application thereof
  • miRNA-340 target gene binding sequence, recombinant plasmid and application thereof
  • miRNA-340 target gene binding sequence, recombinant plasmid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Cloning of Example 1 Mouse IL-17A 3'UTR Nucleic Acid Sequence

[0057] Step S101: The nucleic acid sequence of mmu-IL-17A 3'UTR found in NCBI is from the 535th base to the 1171st base in the IL-17A gene (as shown in SEQ ID No.1), and the designed primers are:

[0058] F: 5'-CGGCTAGCCGACAGAGACCCGCGGCTGACCCCTAAG-3' (as shown in SEQ ID No.2);

[0059] R: 5'-CCGCTCGAGCGGTTTAAAACAGAGTAGGGAGCT-3' (shown in SEQ ID No. 3).

[0060] Step S102: Using mouse genomic DNA as a template, using the nucleotide sequences shown in SEQ ID No.2 and SEQ ID No.3 as primers, using high-fidelity enzymes to perform conventional PCR reaction amplification, and gel after electrophoresis The PCR product was recovered, and NheI and XhoI were used for double digestion, and the digested product was recovered and purified.

Embodiment 2

[0061] Example 2 Construction of the recombinant reporter plasmid of pmir-GLO-IL-17A 3'UTR complete fragment

[0062] Step S201: Transform conventionally prepared DH5α competent cells with the pmir-GLO plasmid for amplification, prepare the pmir-GLO plasmid with a mini-prep kit, check the purity and quality of the plasmid by electrophoresis, perform double digestion of the plasmid with NheI and XhoI, and gel The recovery kit recovers the digested products of large fragments; the pmir-GLO plasmid map is as follows figure 1 As shown, the pmir-GLO plasmid in this embodiment is the pmirGLO Dual-Luciferase miRNA Target Expression vector (abbreviated as pmirGLO).

[0063] Ligation, transformation and clonal amplification of recombinant plasmids

[0064] Step S202: Perform a ligation reaction between the digested mmu-IL-17A 3'UTR PCR product prepared in Example 1 and the pmir-GLO plasmid.

[0065] Step S203: The ligation product is transformed into competent cells DH5α, selectively...

Embodiment 3I

[0067] Example 3 Construction of IL-17A 3'UTR fragment deletion reporter plasmid

[0068] S301: Design the different primers shown in Table 1, and perform PCR using the constructed pmir-GLO-IL-17A 3'UTR complete fragment recombination reporter plasmid in Example 2 as a template to obtain a series of IL-17A deletions of different lengths 3'UTR fragments: F1(+535~+1131), F2(+622~+1131), F3(+759~+1131), F4(+849~+1131), F5(+914~+1131), Wherein, F1 is the 535th base to the 1131st base in the IL-17A gene (as shown in SEQ ID No.4), and F2 is the 622nd to the 1131st base in the IL-17A gene (as shown in SEQ ID No. .5), F3 is the 759th base to the 1131st base in the IL-17A gene (as shown in SEQ ID No.6), and F4 is the 849th to the 1131st base in the IL-17A gene ( As shown in SEQ ID No.7), F5 is the 914th base to the 1131st base in the IL-17A gene (as shown in SEQ ID No.8).

[0069] Table 1 Primers for IL-17A 3'UTR deletion fragment

[0070]

[0071] Step S302: agarose gel electrop...

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 relates to the technical field of molecular biology and regulation of gene expression, in particular to an miRNA-340 target gene binding sequence, a recombinant plasmid and application thereof. The miRNA-340 target gene binding sequence comprises a nucleotide sequence shown as SEQIDNO:1, SEQIDNO:4, SEQIDNO:5, SEQIDNO:6, SEQIDNO:7 or SEQIDNO:19. The miRNA-340 target gene binding sequence can be bonded to miRNA-340, and the constructed recombinant plasmid can quickly, sensitively, easily and conveniently identify whether a target microRNA is bounded to an IL-17A 3'UTR regulating fragment so as to play a regulating role.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and gene expression regulation, in particular to a miRNA-340 target gene binding sequence, a recombinant plasmid and applications thereof. Background technique [0002] MicroRNA (microRNA, miRNA) is a kind of single-stranded small molecule RNA with only 18-25 nucleotides, which widely exists in nematodes, fruit flies, plants and eukaryotes including humans, and has a high degree of conservation. Sexual, tissue-specific and temporal non-coding RNA molecules without an open reading frame (open reading frame). The microRNA gene is located in the intron of the genetically encoded gene, and part of it exists in the intergenic DNA sequence. After forming a mature microRNA, it plays a post-transcriptional role. By complementary pairing with the 3' non-coding region (3'UTR) of the target gene, it can negatively regulate the target gene at the post-transcriptional level, resulting in mRNA trans...

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): C12N15/113C12N15/63C12N1/19C12N5/10C12N15/66C12Q1/6876
CPCC12N15/113C12N15/63C12N15/66C12N2310/14C12Q1/6876
Inventor 阮庆国边江刘芮伶王绍文耿雯雯
Owner SHENZHEN INST OF ADVANCED TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com