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Application 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

Pending Publication Date: 2020-01-03
SHANGHAI GENECHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] There are few reports on DGKZ in tumor-related fields

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Preparation of RNAi lentivirus against human DGKZ gene

[0083] 1. Screening for effective siRNA targets against the human DGKZ gene

[0084] 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.

[0085] Table 1 is targeted at the siRNA target sequence of human DGKZ gene

[0086]

[0087]

[0088] 2. Preparation of lentiviral vector

[0089] Aim at the siRNA target (taking SEQ ID NO: 1 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 2) with Age I and EcoR I restriction endonucleases at both ends; use Age I and EcoR I restriction enzyme Act on the pGCSIL-GFP carrier (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd., figure 1 ), to linearize it, and identify the digested fragments by agarose gel electrophoresis.

[0090]Table 2 Double-stranded DNA Oligo with sticky ends containing Age I and Eco...

Embodiment 2

[0110] Embodiment 2: Real-time fluorescent quantitative RT-PCR method detects the silencing efficiency of DGKZ gene

[0111] 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 inoculated in a 6-well plate 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 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 instruction manual of Promega Company, 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)....

Embodiment 3

[0119] Example 3: Detection of proliferation ability of tumor cells infected with DGKZ-siRNA lentivirus

[0120] 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 inoculated in a 6-well plate 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 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 and read once a day with a Cello...

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Abstract

The invention discloses application of a human DGKZ gene and related drugs thereof. The invention discloses application of the human DGKZ gene in tumor treatment, tumor diagnosis and drug preparation.The invention further constructs a human DGKZ gene small interfering RNA, a human DGKZ gene interfering nucleic acid construct and a human DGKZ gene interfering lentivirus, and discloses applicationsof the siRNA, the interfering nucleic acid construct and the interfering lentivirus. The siRNA or the nucleic acid construct containing the siRNA sequence, and the lentivirus provided by the invention can specifically inhibit the expression of the human DGKZ gene. Especially, the lentivirus can efficiently infect target cells, efficiently inhibit the expression of DGKZ genes in the target cells,further inhibit the growth of tumor cells and promote the apoptosis of the tumor cells, and has important significance in tumor treatment.

Description

[0001] This application is a divisional application of the original application. The filing date of the original application is: 2014-03-19; the application number is: 2014101030095; the title of the invention is: use of human DGKZ gene and related medicines. technical field [0002] The invention relates to the field of biotechnology, and more specifically relates to the use of human DGKZ gene and related medicines. Background technique [0003] 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 (TuschlT, Zamore PD, Sharp PA, Bartel DP.RNAi: double-stranded RNA directs the A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/113C12N15/867C12N7/01A61K31/713A61K48/00A61P35/00C12R1/93
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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