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SiRNA (small interfering ribose nucleic acid) for inhibiting homosapiens chromosome2open reading frame 3(C2 or f3) (GCF) gene expression, carrier of SiRNA for inhibiting GCF gene expression and application

An expression vector and gene expression technology, applied in the field of medical genetic engineering, to achieve the effect of improving radiation sensitivity, benefiting radiotherapy and drug development, and high efficiency of RNA interference

Inactive Publication Date: 2014-06-04
NAT INST FOR RADIOLOGICAL PROTECTION & NUCLEAR SAFETY CHINESE CENT FOR DISEASE CONTROL & PREVENTION +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

GCF can be stimulated by okadaic acid, phorbol lipid, and cyclic AMP, but not by vanadium (Laura B, Hitoshi Y, Ira P and Alfred, Biochemical Characterization of Human GCF Transcription Factor in Tumor Cells, The Journal Biological Chemistry, 1992, Vol.267, No.3, Issue of January25, pp.1689-1694), in addition, it also has some expression in some diseased tissues, for example, the high level expression of GCF mRNA appears in KATO III and AGS (gastric cancer), FEM- In X (melanoma), and U266B1 (myeloma) cells, no expression of GCFmRNA was found in a human fibroblast (W138), a cell line of mice (NIH3T3) and a cell line of monkeys ( CV-1) was not found by cross hybridization

Method used

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  • SiRNA (small interfering ribose nucleic acid) for inhibiting homosapiens chromosome2open reading frame 3(C2 or f3) (GCF) gene expression, carrier of SiRNA for inhibiting GCF gene expression and application
  • SiRNA (small interfering ribose nucleic acid) for inhibiting homosapiens chromosome2open reading frame 3(C2 or f3) (GCF) gene expression, carrier of SiRNA for inhibiting GCF gene expression and application
  • SiRNA (small interfering ribose nucleic acid) for inhibiting homosapiens chromosome2open reading frame 3(C2 or f3) (GCF) gene expression, carrier of SiRNA for inhibiting GCF gene expression and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Screening of SiRNA sequences that inhibit GCF gene expression

[0065] Using network resources and software to conduct bioinformatics analysis on the GCF gene, search NCBI GeneBank to obtain the complete RNA sequence of the GCF gene, numbered NM_003203.4, with a total length of 4455bp, and its coding sequence (CDS, coding sequence) is located at 131-2476. Coding regions were chosen as target sequences for siRNA design. Perform preliminary design and screening on the corresponding siRNA design website (www.ambion.com), and then compare the following design principles to screen out the siRNA sequences. Principles of siRNA sequence design:

[0066] (1) 50-100 bases from the 5'-end of the promoter of the GCF gene.

[0067] (2) Find the 23 bases in the GCF gene sequence, preferably 5'-AA (N 19 )TT-3' (N is any base). If no more than four AAs are found (N 19 ) TT, use AA (N 21 ) make up. If no more than four AAs are found (N 21 ), then use NA (N 21 ) make u...

Embodiment 2

[0118] Embodiment 2 pGPU6 / GFP / Neo-shRNA vector construction

[0119] Including the following steps:

[0120] 1. Annealing of GCF-shRNA template

[0121] (1) GCF-shRNA796, GCF-shRNA1405, GCF-shRNA1696, GCF-shRNA1738 template sequences, positive control hGAPDH-shRNA template sequences and negative control NC-shRNA template sequences were artificially synthesized by Invitrogen, the sense strand or antisense The strand sequence is a hairpin structure.

[0122] (2) Dissolve the synthesized DNA oligo with TE (PH8.0) at a concentration of 100 μM.

[0123] (3) Anneal the sense strand and antisense strand of the hairpin structure to form a complementary double strand: the reaction system is 50 μL, where

[0124] Sense strand (100μM): 5μL; antisense strand (100μM): 5μL; annealing buffer (10×shDNAAnnealing Buffer): 5μL; ddH 2 O: 35 μL.

[0125] Perform annealing treatment on the PCR instrument according to the following procedures: 95°C, 5min; 85°C, 5min; 75°C, 5min; 70°C, 5min; St...

Embodiment 3

[0144] Example 3 Establishment and screening of pGPU6 / GFP / Neo-shRNA transfected HeLa cell line

[0145] pGPU6 / GFP / Neo-shRNA796 (named plasmid No. 1), pGPU6 / GFP / Neo-shRNA1405 (named plasmid No. 2), pGPU6 / GFP / Neo-shRNA1696 (named plasmid No. 3) constructed in Example 2 , pGPU6 / GFP / Neo-shRNA1738 (named plasmid No. 4), positive control pGPU6 / GFP / Neo-hGAPDH-shRNA (named plasmid No. 5), and negative control pGPU6 / GFP / Neo-NC-shRNA (named plasmid 6 No. plasmid) was transfected into human normal Hela cells to establish a monoclonal cell line, the specific steps are as follows:

[0146] 1. Determine the lethal dose of G418 for HeLa (normal human Hela cells were purchased from the Fourth Laboratory of the Second Academy of Military Medical Sciences)

[0147] The pGPU6 / GFP / Neo vector was purchased from Shanghai Gemma Company. It contains the anti-G418 gene and expresses green fluorescent protein. Therefore, these two indicators are used to screen successfully transfected monoclonal cells...

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Abstract

The invention discloses a SiRNA (small interfering ribose nucleic acid) for inhibiting homosapiens chromosome 2 open reading frame 3(C2 or f3) (GCF) gene expression, which belongs to the field of medical genetic engineering. Deoxyribose nucleic acid (DNA) for an encoding SiRNA is selected from arbitrary group of (a), (b) and (c): (a) a base sequence of a positive-sense strand is shown as SEQ ID No.1, and the base sequence of an antisense strand is shown as SEQ ID No.2; (b) the base sequence of the positive-sense strand is shown as SEQ ID No.3, and the base sequence of the antisense strand is shown as SEQ ID No.4; and (c) the base sequence of the positive-sense strand is shown as SEQ ID No.5, and the base sequence of the antisense strand is shown as SEQ ID No.6. The invention further discloses a recombinant plasmid which comprises the DNA of the encoding SiRNA and a GCF-shRNA-HeLaCGMCC (China general microbiological culture collection center) No.5821, and a cell line has high RNA interfering efficiency, strong specificity and no toxic and side effects and can effectively inhibit GCF gene expression. The SiRNA for inhibiting GCF gene expression, the recombinant plasmid and the GCF-shRNA-HeLa monoclonal cell can also be used for preparing medicines for treating cervical cancer.

Description

technical field [0001] The invention relates to the field of medical genetic engineering. Specifically, it relates to a siRNA for inhibiting GCF gene expression, its carrier and application. Background technique [0002] RNA interference (RNA interference, RNAi) refers to the specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA (dsRNA), resulting in the silence of target gene expression and the loss of corresponding functional phenotypes Phenomenon. RNAi is a conserved defense mechanism from lower organisms to mammals. The double-stranded RNA is cut into small fragments of about 19-22nt interfering RNA (siRNA) by Dicer enzyme in the cell to form a siRNA-protein complex (siRNP). SiRNA recognizes and degrades mRNA with homologous sequences and finally leads to specific Gene silencing (Liu JY et al., RNAi-gene silencing mediated by dsRNAs, Section Genet Foreign Med Sci, 2004, 27(1):4-10). Therefore, RNA interference technology h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N15/85C12N5/10A61K48/00A61P35/00
Inventor 丁库克崔巍苟巧周平坤苏旭杨川杰王春英尹玲玲崔庆佳刘建香武云云刘晓丹张士猛王春燕董凌月齐雪松崔宏星刘青杰张庆召尚兵毛玲刘淑娟
Owner NAT INST FOR RADIOLOGICAL PROTECTION & NUCLEAR SAFETY CHINESE CENT FOR DISEASE CONTROL & PREVENTION
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