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SPOP (Speckle-Type Pox Virus Zinc Finger Protein) and mutant expressed C4-2 stable cell line and establishment method

A method of constructing mutants, applied to cells modified by introducing foreign genetic material, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as lack of understanding and not very thorough

Inactive Publication Date: 2018-06-29
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, the research on its mechanism is not very thorough, what kind of changes SPOP mutation will cause in prostate cancer, the current understanding is still relatively little

Method used

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  • SPOP (Speckle-Type Pox Virus Zinc Finger Protein) and mutant expressed C4-2 stable cell line and establishment method
  • SPOP (Speckle-Type Pox Virus Zinc Finger Protein) and mutant expressed C4-2 stable cell line and establishment method
  • SPOP (Speckle-Type Pox Virus Zinc Finger Protein) and mutant expressed C4-2 stable cell line and establishment method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0033] Example 1: Construction of SPOP wild-type and mutant 1 and mutant 2 plasmids based on Ploc.rfp vector

[0034] (1) PCR method was used to amplify SPOP wild type and its mutant 1 and mutant 2 genes: SPOP-5': GAC ATCGAT TAC AAG GAT GAC GAT GAC AAG ATG TCA AGG GTT CCA AGT CCT CCAC

[0035]SPOP-3':CGGCT AGC TTA GGA TTG CTT CAG GCG TTT G

[0036] (2) The PCR product obtained in step (1) is detected by electrophoresis, and the next step of PCR reaction is carried out;

[0037] (3) Dilute the product 100 times, add SPOP-3' and FLAG-5' primers, and add 3×FLAG tags to the SPOP wild type and its mutant 1 and mutant 2 sequences. And run the gel, cut off the correct band, and recover it with TaKaRa miniBEST Agarose Gel DNA Extraction Kit;

[0038] FLAG-5': GCGGCCGC ATG GAC TAC AAA GAC CAT GAC GGT GAT TAT AAA GAT CATGAC ATC GAT TAC

[0039] (4) The vector, the constructed SPOP fragment and Ploc.rfp were subjected to a double enzyme digestion reaction with Not I and Nhe I, respect...

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Abstract

The invention relates to the field of biomedical research and establishes an SPOP (Speckle-Type Pox Virus Zinc Finger Protein) and mutant expressed prostatic cancer C4-2 cell line. Through primer synthesis, PCRs (Polymerase Chain Reactions) and enzyme digestion reactions, a wild SPOP as well as a mutant 1 gene and a mutant 2 gene thereof are respectively and successfully connected with a Ploc.rfpvector. The accuracy of plasmid is verified through the enzyme digestion reactions and sequencing. Furthermore, by using a method that lentivirus is established in HEK293T cells, a cell culture liquidwith the lentivirus is collected to infect prostatic cancer C4-2 cells, and through 12-16 days of 8-12mu g / mL Blasticidin screening, a C4-2 cell line with high expression of the wild SPOP as well asthe mutant 1 gene and the mutant 2 gene thereof is obtained. The invention provides a study system for studying metabolic disorders caused by SPOP mutation of prostatic cancer.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a C4-2 stable cell line for constructing SPOP wild type and its mutant expression and a construction method thereof. Background technique [0002] SPOP is currently known as the gene with the highest mutation rate in prostate cancer. The molecular weight of SPOP protein is 42kDa. Mutations present in about 6-15% of prostate cancer patients 1 . SPOP protein is an E3 ubiquitination ligase that can mediate the ubiquitination degradation of some proteins. In prostate cancer, the SPOP mutation sites are mainly concentrated in the substrate-binding MATH domain, and its mutation will cause the protein that was originally degraded by binding to it to be ubiquitinated to no longer be degraded by ubiquitination, resulting in the accumulation of oncoproteins in the cell. The currently reported SPOP substrates are AR 2 、ERG 3,4 、PTEN 5 、DEK 6 Wait. The study found that in prostate cancer,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/66
CPCC12N9/93C12N15/66C12N15/86C12N2740/15043C12Y603/02019
Inventor 朴海龙颜敏
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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