Fluorescent reporter system for screening and identification of circular RNA translation proteins and its construction

A technology for reporting systems and construction methods, applied in recombinant DNA technology, biochemical equipment and methods, and the use of vectors to introduce foreign genetic materials, etc., can solve the problem of lack of tools and methods for large-scale screening of circular RNA translation proteins, etc. problem, to achieve the effect of solving the screening problem

Active Publication Date: 2019-03-05
GUANGZHOU GENESEED BIOTECH
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Problems solved by technology

[0003] At present, the specific function of circular RNA has not yet been clarified, and there are only several assumptions: (1) circular RNA can act as a "sponge" (sponge) to absorb miRNA and inhibit its function; Pairing directly regulates the level of other RNAs; (3) circular RNAs can bind to proteins, inhibit protein activity, recruit components of protein complexes or regulate protein activity; some researchers have also reported that circular RNAs can also serve as templates for translation to guide proteins There are many reports on viruses and artificially synthesized circular RNA translation proteins; there are few discoveries on circular RNA translation proteins in human and other higher organisms. The reason may be that the previous research on circular RNAs was not known In addition, there is a lack of effective tools and methods for large-scale screening of circular RNA translation proteins

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  • Fluorescent reporter system for screening and identification of circular RNA translation proteins and its construction
  • Fluorescent reporter system for screening and identification of circular RNA translation proteins and its construction
  • Fluorescent reporter system for screening and identification of circular RNA translation proteins and its construction

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Embodiment approach

[0030] Concrete implementation scheme is as follows:

[0031] After synthesizing the target framework nucleotide sequence SEQ ID NO: 1 (Shanghai Jierui Biological Co., Ltd.), the PCR amplification method was used to introduce the NheI and XhoI restriction site sequences when designing primers. The sequences of the designed primers were:

[0032] primer-F0: 5'-GCGCTAGCGAGTTTCTAAAATTAAACTA-3' (SEQ ID NO: 2)

[0033] primer-R0: 5'-CGCTCGAGCTCTAAAATTATTCGTTCATGGCTT-3' (SEQ ID NO: 3)

[0034] The reaction system of PCR amplification is as follows: PCR is 30 μl total system, specifically 2×PCR MIX 15 μl, 10 mM upstream and downstream primers 1.5 μl each, chemically synthesized gene frame SEQ ID NO.1 template 1 μl, supplemented with sterilized deionized water 30 μl system; the reaction conditions are: 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 62°C for 30 seconds, and extension at 72°C for 2 minutes, a total of 30 cycles. After the PCR reaction cycle, cont...

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Abstract

The invention provides a fluorescence reporting system for screening and identifying circRNA (circular ribonucleic acid) translation proteins. A nucleotide sequence of an expression cassette of the fluorescence reporting system is shown as SEQ ID NO.1. The invention further provides a construction method of the fluorescence reporting system. The fluorescence reporting system can screen and identify the circRNA translation proteins effectively.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a fluorescent reporter system for screening and identifying circular RNA translation proteins and a construction method thereof. Background technique [0002] Circular RNA (circular RNA, circRNA) is a type of RNA molecule with special functions that is different from traditional linear RNA. Most circRNAs are composed of exon sequences, which are conserved in different species and have expression specificity in tissues and different developmental stages. With the development of deep RNA sequencing and large-scale bioinformatics technology, researchers have discovered that there are a large number of circularized RNA molecules in organisms, and circularized RNAs are very stable in organisms because they form closed circles. Because circular RNA is insensitive to nucleases, it is more stable than linear RNA, which makes circular RNA have obvious advantages in the develo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/65
CPCC12N15/65C12N15/66C12N15/85C12N2800/107
Inventor 张茂雷刘明李自强薛权灿
Owner GUANGZHOU GENESEED BIOTECH
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