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Application and related pharmaceuticals of human ubiquitin-protein ligase 138 (RNF138) gene

A gene and drug technology, applied in the use of human RNF138 gene and related drug fields, can solve the problems to be further studied and so on

Active Publication Date: 2013-09-18
SHANGHAI JI KAI GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, studies have found that overexpressing exogenous RNF138 gene in Hela cells can enhance the ability of cell colony formation, but the mechanism of RNF138 remains to be further studied (Tucker W.A, Christopher W, William S.B. The E3ubiquition ligase NARF promotes colony formation in vitro and exhibits enhanced expression levels in glioblastoma multiforme in vivo. American J Undergraduate research 2010, 9: 23-30)

Method used

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  • Application and related pharmaceuticals of human ubiquitin-protein ligase 138 (RNF138) gene
  • Application and related pharmaceuticals of human ubiquitin-protein ligase 138 (RNF138) gene
  • Application and related pharmaceuticals of human ubiquitin-protein ligase 138 (RNF138) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1: Preparation of RNAi lentivirus for human RNF138 gene

[0072] 1. Screening effective siRNA targets against human RNF138 gene

[0073] Select the coding region sequence of the human RNF138 (NM 016271) gene from Genbank, and obtain a sequence of 21 bases starting from every other base; use the design software of Shanghai Jikai Gene Chemical Technology Co., Ltd., and use the human RNF138 mRNA sequence as a template , to determine 13 effective siRNA target sequences (SEQ ID NO: 1-13), as shown in Table 1:

[0074] Table 1 is targeted at the siRNA target sequence of human RNF138 gene

[0075]

[0076] Aim at the siRNA target (take SEQ ID NO: 8 as an example) to synthesize a double-stranded DNA Oligo sequence (see Table 2) with Age I and EcoR I restriction sites at both ends; The enzyme acts on the pGCSIL-GFP carrier (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to make it linearized (the reaction system is shown in Table 4, 37° ...

Embodiment 2

[0098] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of RNF138 gene

[0099] Human pancreatic cancer Panc-1 cells in the logarithmic growth phase were trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI value: 10), an appropriate amount of the lentivirus prepared in Example 1 was added, the culture medium was replaced after 24 hours of cultivation, and the cells were collected after the infection time reached 5 days. Total RNA was extracted according to the instruction manual of Invitrogen's Trizol. According to the M-MLV operation manual of Promega Company, RNA was reverse-transcribed to obtain cDNA (see Table 7 for the reverse transcription reaction system), reacted at 42°C for 1h, and then bathed in a water bath at 70°C for 10min to inactivate the reverse tr...

Embodiment 3

[0107] Example 3: Detection of proliferation ability of tumor cells infected with siRNF138-Lentivims lentivirus

[0108] Human pancreatic cancer Panc-1 cells in the logarithmic growth phase were digested with trypsin to make a cell suspension (the number of cells was about 5×10 4 / ml) were inoculated in a 6-well plate and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI value is 10), an appropriate amount of virus was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 5 days, the cells of each experimental group in the logarithmic growth phase were collected. The complete medium was resuspended into a cell suspension (2×10 4 / ml), inoculate a 96-well plate at a cell density of about 2000 / well. 5 replicate wells in each group, 100 μl per well. After laying the board, place at 37°C, 5% CO 2 Incubator cultivation. From the second day after plating, the plate was detected ...

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Abstract

The invention discloses an application related pharmaceuticals of a human ubiquitin-protein ligase 138 (RNF138) gene and particularly discloses an application of the human RNF138 gene in tumor treatment and pharmaceutical preparation. The invention further structures an oligonucleotide molecule, a human RNF138 gene interference lentiviral vector and a human RNF138 gene interference lentivirus for human RNF138 gene separation and discloses applications thereof. The oligonucleotide molecule or the lentiviral vector and the lentivirus containing an oligonucleotide molecule sequence provided by the invention can specifically inhibit the human RNF138 gene expression, and particularly, the lentivirus can efficiently infect target cells, effectively inhibit the RNF138 gene expression of the target cells, further inhibit the tumor cell growth, promote the tumor cell apoptosis and have an important meaning on tumor treatment.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically relates to the use of human RNF138 gene and related medicines. Background technique [0002] RING finger proteins are a class of zinc finger proteins that contain a ring domain, a zinc finger structure consisting of 40–60 amino acids that can bind two zinc atoms (Saurin AJ, Borden KL, Boddy MN, et al.Does this have a familiar RNG.Trends Biochem Sci 1996,21:208-214.Freemont PS The RNG finger Anovel protein sequence motif related to the zinc finger.Ann N Y Acad Sci,1993,684:174-192.Borden KL, Freemont PS. The RNG finger domain: a recent example of a sequence-structure family. Curr Opin Struct Biol, 1996, 6: 395-401). This domain may be involved in protein-protein interactions, and it is often involved in the degradation of misfolded proteins and proteins that have completed their mission in the cell. This process is an important quality control process in cells and is ve...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/113C12N15/867A61K48/00A61P35/00C12N15/11
Inventor 朱向莹孙琴高博谢胜华金杨晟瞿红花曹跃琼
Owner SHANGHAI JI KAI GENE TECH CO LTD
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