Use of human zfx gene and related medicines

A gene and drug technology, applied in the application of human ZFX gene and its related drug fields

Active Publication Date: 2011-12-14
SHANGHAI GENECHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, the reports of ZFX gene in tumor-related fields are still blank.

Method used

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  • Use of human zfx gene and related medicines
  • Use of human zfx gene and related medicines
  • Use of human zfx gene and related medicines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1: Preparation of RNAi lentivirus against human ZFX gene

[0074] 1. Screening for effective siRNA targets against the human ZFX gene

[0075] Retrieve ZFX (NM_003410.2) gene information from Genbank; design effective siRNA targets for ZFX gene by using the design software Genechem of Shanghai Jikai Gene Chemical Technology Co., Ltd. In the coding sequence (CDS) region of the ZFX gene, a sequence of 21 bases was obtained every other base, and Table 1 lists 42 effective siRNA target sequences for the ZFX gene.

[0076] Table 1 is targeted at the siRNA target sequence of human ZFX gene

[0077]

[0078]

[0079]

[0080] Aim at the siRNA target (taking SEQ ID NO: 19 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 2) containing AgeI and EcoRI restriction sites at both ends; act on pGCSIL- GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to linearize it, and identify the digested fragments by...

Embodiment 2

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

[0099] Human laryngeal cancer Hep 2 cells, lung cancer 95D cells, gastric cancer AGS cells, liver cancer SMMC-7721 cells, breast cancer MCF-7 cells and glioma U251 cells in logarithmic growth phase were digested with trypsin to make cell suspension ( The number of cells is 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, Hep2: 10, 95D: 2, AGS: 100, SMMC-7721: 20, MCF-7: 20, U251: 10) value, add an appropriate amount of the virus prepared in Example 1 and incubate for 24 hours Afterwards, the medium was replaced, 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 instruction manual of Promega Company, the RNA was reverse-transcr...

Embodiment 3

[0106] Example 3: Detection of proliferation ability of tumor cells infected with ZFX-siRNA lentivirus

[0107] Human laryngeal cancer Hep 2 cells, lung cancer 95D cells, gastric cancer AGS cells, liver cancer SMMC-7721 cells, breast cancer MCF-7 cells and glioma U251 cells in logarithmic growth phase were digested with trypsin to make cell suspension ( The number of cells is 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, Hep2: 10, 95D: 2, AGS: 100, SMMC-7721: 20, MCF-7: 20, U251: 10), an appropriate amount of virus was added, and the culture medium was replaced after 24 hours. 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 g...

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PUM

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Abstract

The invention discloses the use of human ZFX gene and related medicines. The invention discloses the application of human ZFX gene in tumor treatment, tumor diagnosis and medicine preparation. The present invention further constructs human ZFX gene small molecule interference RNA, human ZFX gene interference nucleic acid construct, human ZFX gene interference lentivirus and discloses their applications. The siRNA provided by the present invention or the nucleic acid construct containing the siRNA sequence, and the lentivirus can specifically inhibit the expression of the human ZFX gene, especially the lentivirus, which can efficiently infect target cells and efficiently inhibit the expression of the ZFX gene in the target cells , and then inhibit the growth of tumor cells and promote the apoptosis of tumor cells, which is of great significance in tumor therapy.

Description

technical field [0001] The present invention relates to the field of biotechnology, and more specifically relates to the use of human ZFX gene and related medicines. Background technique [0002] The phenomenon of ribonucleic acid interference (RNA interference, RNAi) refers to the specific degradation of the mRNA when a double-stranded RNA (dsRNA) homologous to a certain sequence of the endogenous mRNA coding region is introduced into the cell, resulting in the silencing of the gene expression . Studies have shown that double-stranded RNA with a length of 21-23nt can specifically cause RNAi at the transcriptional and post-transcriptional levels (Tuschl T, Zamore PD, Sharp PA, Bartel DP.RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000; 101: 25-33.). Tumor is a major disease that threatens human health. Although cancer patients have undergone chemotherapy, radiotherapy and comprehensive treatment, their five-y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/113C12N15/867A61K48/00A61P35/00
Inventor 曹跃琼吕大龙朱向莹韩海雄瞿红花金杨晟
Owner SHANGHAI GENECHEM
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