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Applications of human DGKZ gene and related drugs thereof

A gene and drug technology, applied in the field of the use of human DGKZ gene and related drugs, can solve the problem of few reports on DGKZ

Inactive Publication Date: 2015-09-23
SHANGHAI GENECHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There are few reports on DGKZ in tumor-related fields

Method used

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  • Applications of human DGKZ gene and related drugs thereof
  • Applications of human DGKZ gene and related drugs thereof
  • Applications of human DGKZ gene and related drugs thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Preparation of RNAi lentivirus against human DGKZ gene

[0082] 1. Screening for effective siRNA targets against the human DGKZ gene

[0083] Retrieve DGKZ (NM_003646.3) gene information from Genbank; design effective siRNA targets for DGKZ gene. Table 1 lists 13 effective siRNA target sequences for the DGKZ gene.

[0084] Table 1 is targeted at the siRNA target sequence of human DGKZ gene

[0085]

[0086]

[0087] 2. Preparation of lentiviral vector

[0088] For the siRNA target (take SEQ ID NO: 1 as an example), synthesize a double-stranded DNA Oligo sequence containing Age I and EcoR I restriction sites at both ends (Table 2); use Age I and EcoR I restriction endonucleases Act on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to linearize it, and identify the digested fragments by agarose gel electrophoresis.

[0089]Table 2 Double-stranded DNA Oligo with sticky ends containing Age I and EcoR I...

Embodiment 2

[0109] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of DGKZ gene

[0110] The human glioma U87 cells in the logarithmic growth phase were trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml) were seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, U87:10) value, an appropriate amount of the virus prepared in Example 1 was added, the culture medium was replaced after 24 hours of culture, 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 Promega's M-MLV operating instructions, RNA was reverse-transcribed to obtain cDNA (see Table 7 for the reverse transcription reaction system, react at 42 °C for 1 h, and then bathe in a water bath at 70 °C for 10 min to inactivate reverse transcriptase).

[0111] Real-time...

Embodiment 3

[0118] Example 3: Detection of proliferation ability of tumor cells infected with DGKZ-siRNA lentivirus

[0119] The human glioma U87 cells in the logarithmic growth phase were trypsinized to make a cell suspension (the number of cells was about 5×10 4 / ml) were seeded in 6-well plates and cultured until the cell confluency reached about 30%. According to the multiplicity of infection (MOI, U87:10), 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 in 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 cells / 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 and read once a day with a Cell...

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Abstract

The invention discloses applications of human DGKZ gene and related drugs thereof. Applications of human DGKZ gene in tumor treatment, tumor diagnosis, and drug preparation are disclosed. The invention further constructs human DGKZ gene micromolecule interference RNA, human DGKZ gene interference nucleic acid construct, and human DGKZ gene interference slow virus, and discloses the applications thereof. The provided siRNA or nucleic acid construct containing the siRNA and slow virus can inhibit the expression of human DGKZ gene specifically, especially the slow virus can infect target cells efficiently and inhibit the expression of DGKZ gene in target cells efficiently, thus the tumor cell growth is inhibited, apoptosis of tumor cells is promoted, and thus the provided siRNA, nucleic acid construct, and slow virus have an important meaning in tumor treatment.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to the use of human DGKZ 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 at21to23nucleotide 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-year survival ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/113C12N15/867C12N7/01A61K48/00A61P35/00
Inventor 朱向莹孙琴高博张晓慧金杨晟瞿红花曹跃琼
Owner SHANGHAI GENECHEM
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