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siRNA, recombinant vector and application of silencing human ran gene

A recombinant vector and silencing technology, applied in the field of molecular biology and biopharmaceuticals, can solve the problem that mRNA cannot be translated into protein

Active Publication Date: 2018-09-25
CHINA INST FOR RADIATION PROTECTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When delivered into the cell, the hairpin sequence is expressed to form a double-stranded RNA (shRNA), which is processed by the RNAi channel and then bound to the RNA-induced silencing complex (RISC), which The complex can bind and cleave the mRNA that matches the siRNA sequence, resulting in the inability of the mRNA to be translated into the corresponding protein, and finally silencing the expression of the corresponding protein in the cell

Method used

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  • siRNA, recombinant vector and application of silencing human ran gene
  • siRNA, recombinant vector and application of silencing human ran gene
  • siRNA, recombinant vector and application of silencing human ran gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] This example is used to illustrate the construction method of siRNA for silencing human RAN gene provided by the present invention.

[0019] According to the nucleotide sequence of the RAN gene reported in Genbank, five siRNA target sequences that inhibit the expression of the RAN gene were designed with reference to the siRNA design principles, respectively SEQ ID NO: 1-5, and a random control sequence SEQ ID NO.6 was designed at the same time ,As follows:

[0020] SEQ ID NO. 1: TAAGTCGTGCTCATACTGT;

[0021] SEQ ID NO. 2: GTCATTTGACTGGTGAATT;

[0022] SEQ ID NO. 3: GTTCAAACTTGTATTGGTT;

[0023] SEQ ID NO. 4: CCCTAACTTGGAATTTGTT;

[0024] SEQ ID NO.5: GGATATTAAGGACAGGAAA;

[0025] SEQ ID NO. 6: TTCTCCGAACGTGTCACGT.

[0026] According to Blast gene homology analysis, the sequence SEQ ID NO.1-5 is not homologous to any human non-RAN gene sequence, so as to ensure the specificity of gene suppression.

[0027] For the above target sequences, referring to the siRNA des...

Embodiment 2

[0059] This example is used to illustrate the construction method of the siRNA recombinant vector for silencing human RAN gene provided by the present invention.

[0060] The vector used was the lentiviral vector pGC-LV (purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.). Its frame structure is:

[0061] hU6-MCS-Ubiquitin-EGFP-IRES-puromycin.

[0062] The construction method of the recombinant vector is as follows:

[0063] Put the sense strand and antisense strand of the 5 pairs of shRNA expression framework DNA sequence SEQ ID NO.1'-5' of the siRNA in Example 1 in an annealing buffer (10mM Tris-HCl, 1mM EDTA, 0.1mMNaCl) in a 90°C water bath 15min, then naturally cool to room temperature, react to synthesize double-stranded DNA (dsDNA) sequence, digest the double-stranded DNA sequence with EcoRI and AgeI, and use EcoRI and AgeI to digest the lentiviral vector pGC-LV at the same time to produce a double-stranded DNA sequence Using T4 DNA ligase to ligate the ...

Embodiment 3

[0066] This example is used to illustrate the inhibitory effect of the siRNA recombinant vector for silencing human RAN gene provided by the present invention on the expression of RAN gene in human hepatocyte HL-7702.

[0067] 1. Cell culture

[0068] The normal human liver cells HL-7702 in good condition were inoculated in 6-well cell culture plates in RPMI1640 medium containing 20% ​​(v / v) calf serum, 100 U / ml penicillin and 100 mg / L streptomycin ( USA, Invitrogen Company), at 37°C, 5% (v / v) CO 2 cultured under the same conditions until the cell confluency reached about 80%. At the same time, the cultured cells were divided into 6 groups, namely 5 experimental groups and 1 control group.

[0069] 2. Gene transfection

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Abstract

The invention relates to siRNA silencing a human RAN gene. The siRNA has any sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5. The invention further provides a recombinant vector with the siRNA silencing the human RAN gene, and application of the siRNA silencing the human RAN gene and the recombinant vector. By adopting the siRNA silencing the human RAN gene and the recombinant vector, an effective technique tool can be provided for study on functions of the RAN gene.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biopharmaceuticals, and specifically relates to siRNA for silencing human RAN gene, a recombinant vector and its application. Background technique [0002] RNAi is a post-transcriptional gene silencing mechanism that reduces the expression of target genes by causing homologous mRNA degradation by double-stranded RNA (dsRNA). This phenomenon is conserved and widespread during evolution. At present, RNAi technology has entered many fields of biological science research and has become a major biological research tool. It provides a new research method for the analysis of gene function, signal transduction pathway and gene therapy. It won the Nobel Prize in 2006. prize. RNAi has the following characteristics and advantages: 1. The specificity of action, it only produces inhibitory effect on its homologous mRNA, and base mismatch will greatly reduce the inhibitory efficiency; 2. RNAi is...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/867C12N15/63C12N5/10
Inventor 党旭红左雅慧刘建功张慧芳刘占旗段志凯刘红艳杨彪王超
Owner CHINA INST FOR RADIATION PROTECTION
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