Use of human TNFRSF12A gene and related drugs

A gene and drug technology, applied in the application of human TNFRSF12A gene and related drugs, can solve the problem of few reports of TNFRSF12A

Pending Publication Date: 2016-07-20
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] There are few reports on TNFRSF12A in tumor-related fields

Method used

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  • Use of human TNFRSF12A gene and related drugs
  • Use of human TNFRSF12A gene and related drugs
  • Use of human TNFRSF12A gene and related drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Preparation of RNAi lentivirus for human TNFRSF12A gene

[0081] 1. Screening for effective siRNA targets against human TNFRSF12A gene

[0082] Retrieve TNFRSF12A gene information from Genbank; design effective siRNA targets for TNFRSF12A gene. Table 1 lists the effective siRNA target sequences for the TNFRSF12A gene.

[0083] Table 1 is targeted at the siRNA target sequence of human TNFRSF12A gene

[0084]

[0085] 2. Preparation of lentiviral vector

[0086] Aiming at the siRNA target (taking SEQIDNO: 1 as an example) synthetic double-stranded DNA Oligo sequence (table 2) containing AgeI and EcoRI restriction endonuclease at both ends; Acting 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.

[0087] Table 2 Double-stranded DNA Oligo with sticky ends containing AgeI and EcoRI restriction sites at both ends

[0088]...

Embodiment 2

[0107] Example 2 Real-time fluorescent quantitative RT-PCR method to detect the silencing efficiency of TNFRSF12A gene

[0108] Human liver cancer SMMC-7721 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, SMMC-7721:10) 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 4 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 inactiv...

Embodiment 3

[0115] Example 3 Detection of proliferation ability of tumor cells infected with TNFRSF12A-siRNA lentivirus

[0116] Human liver cancer SMMC-7721 in the logarithmic growth phase was 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, SMMC-7721:10), an appropriate amount of virus was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 4 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 on...

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Abstract

The invention discloses the use of a human TNFRSF12A gene and related drugs. The invention discloses the use of the human TNFRSF12A gene in tumor therapy, tumor diagnosis and drug preparation. The invention further constructs human TNFRSF12A gene small interfering RNA, a human TNFRSF12A gene interference nucleic acid construct, and a human TNFRSF12A gene interference slow virus and discloses their use. The siRNA or the nucleic acid construct containing the siRNA sequence, and the slow virus provided by the invention can specifically inhibit the expression of the TNFRSF12A gene, especially the slow virus can efficiently infect target cells, effectively inhibit the expression of the TNFRSF12A gene in the target cells, and therefore inhibits the growth of tumor cells and promotes tumor cell apoptosis, thus having important significance in tumor treatment.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to the use of human TNFRSF12A gene and related medicines. Background technique [0002] RNA interference (RNAi) is short double-stranded RNA (dsRNA) composed of nucleotides for post-transcriptional gene silencing. It can efficiently and specifically block the expression of a specific gene in the body, leading to its degradation, thereby causing the silencing of a specific gene in the organism, and causing the cells to show the absence of a certain gene phenotype. It is a commonly used research gene emerging in recent years. Functional, laboratory techniques for finding cures for disease. 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, ZamorePD, SharpPA, BartelDP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21to23nucleotide interva...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/113C12N15/867A61K31/713A61P35/00
Inventor 聂勇战吴开春董加强胡思隽窦建华唐光波吴健
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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