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Use of DLP in Pre-mRNA splicing and cell cycle control

A DLP, applied technology, applied in the field of DLP protein

Inactive Publication Date: 2008-01-09
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the emergence of many new proteins not previously associated with splicing is surprising

Method used

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  • Use of DLP in Pre-mRNA splicing and cell cycle control
  • Use of DLP in Pre-mRNA splicing and cell cycle control
  • Use of DLP in Pre-mRNA splicing and cell cycle control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Extraction of total mRNA

[0118] Extraction of total cellular mRNA using Trizol from Invitrogen TM reagent. Take 1×10 each 7 MCF-7 cells, HepG2 and Ishikawa cells were collected by centrifugation, and 1ml Trizol was added TM Reagent, blow repeatedly to break the cells, add 0.2ml chloroform after standing still at room temperature for 5 minutes, shake vigorously for 15 seconds, 12000rpm at 4°C for 15 minutes, collect supernatant and precipitate with isopropanol, wash once with 70% ethanol, and dry at room temperature. RNA was resuspended in DEPC-treated H 2 In O, it was used for cDNA first-strand synthesis after quantification by spectrophotometer.

[0119] cDNA duplex synthesis

[0120] Using NEB's ProtoScript TM First Strand cDNA Synthesis Kit uses the mRNA mentioned in 2.3 as a template to synthesize the first and second strands of cDNA. The brief description is as follows: 1ng-2ugmRNA, 2ul Primer dT23VN, 4ul dNTP, add H 2 0 to 16ul reaction system, 70°C for...

Embodiment 2

[0149] Preparation of anti-GST-DLP polyclonal antibody and Western blot hybridization

[0150] The purified GST-DLP fusion protein was used to immunize New Zealand white rabbits to prepare polyclonal antibodies against DLP. For Western blot, the extracted protein was quantified using Bio-Rad's DC protein quantification reagent, and then subjected to 15% SDS-PAGE electrophoresis. After electrophoresis, the protein was transferred to a nitrocellulose membrane. After the membrane was blocked with 5% skimmed milk powder for 2 hours, a 1:30 dilution of rabbit anti-DLP polyclonal antibody or a 1:5000 dilution of anti-c-myc monoclonal antibody (Invitrogen) was added at 4°C overnight, and washed three times with TBST After) (10 minutes / time), add horseradish enzyme-labeled anti-rabbit IgG (Amersham Pharmacia Biotech) or goat anti-mouse IgG (Santa Cruz) diluted 1:4000, incubate for 1 hour, and wash the membrane three times with TBS Add substrate solution for color development.

Embodiment 3

[0152] GST Pull-down

[0153] Using the TNT Rabbit Reticulocyte lysate and TNT T7polymerase labels kit from Promega, pcDNA-Prp6, hnRNPF, PQBP and APC4 were transcribed and translated in vitro. Proceed as follows:

[0154] (1) Add the reagents to the 1.5ml tube in the following order

[0155] 25ul TNT Lysate

[0156]2ul TNT reaction buffer

[0157] 1ul TNT RNA polymerase

[0158] 1ul Amino Acid Mix, minus Met(1mM)

[0159] 2ul [ 35 S] Methionine (>1000ci / mmol)

[0160] 1ul Rnasin Ribonuclease inhibitor

[0161] 2ul pcDNA-Prp6, hnRNPF, PQBP and APC4

[0162] 16ul nuclease-free water

[0163]

[0164] 50ul total volume

[0165] 30°C for 90 minutes.

[0166] (2) Add 50ul glutathione agarose beads, 500ng-10ug GST or GST-DLP, and 4-5ul in vitro transcription and translation products into a 1.5ml tube.

[0167] (3) Incubate at 4°C for 2 hours, 13000rpm for 2 minutes, discard the supernatant. Wash glutathione agarose beads ...

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Abstract

The invention relates to DLP protein, gene codzing DLP, and the application of DLP in preparation of splicing and medicine relating to agent of Pre-mRNA and adjustment of cell period, also, it relates to the application designed for DLP in RNAi preparing medicine against tumber.

Description

field of invention [0001] The present invention relates to the DLP protein, the gene encoding DLP, the application of DLP in the preparation of reagents and medicines related to Pre-mRNA splicing and cell cycle regulation, and the present invention also relates to the use of RNAi (RNA interference) designed for DLP in the preparation of drugs for treating tumors Uses in medicine. Background technique [0002] Gene expression in eukaryotic cells is a multi-step complex process, including initiation, elongation and termination of transcription. During the transcription process, the primary Pre-mRNA undergoes capping at the 5' end, intron excision, exon joining, cleavage at the 3' end, and addition of polyadenylic acid to form mature mRNA, which is transported to the cytoplasm for translation into protein. The size of the proteome in an organism not only comes from the size of the number of genes, but also depends on the diversity of proteins. The research on the mechanism o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K16/18C12N15/12C12N15/63A61K48/00A61P35/00
Inventor 尚永丰沈岩孙晓静
Owner PEKING UNIV
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