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Construction of human RNF20 gene overexpression plasmid vector and effect of human RNF20 gene overexpression plasmid vector on inhibiting cancer cells

A technology of gene overexpression and plasmid vectors, applied in vectors, nucleic acid vectors, DNA/RNA fragments, etc., to achieve the effects of promoting apoptosis, promoting transcription and translation, and inhibiting proliferation and migration

Active Publication Date: 2021-01-26
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no relevant report on the construction of human RNF20 gene overexpression vector. The present invention constructs a human RNF20 gene overexpression plasmid vector through molecular related technology, and finds that it can inhibit the proliferation and migration of cancer cells and promote the apoptosis of cancer cells. play an important role in

Method used

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  • Construction of human RNF20 gene overexpression plasmid vector and effect of human RNF20 gene overexpression plasmid vector on inhibiting cancer cells
  • Construction of human RNF20 gene overexpression plasmid vector and effect of human RNF20 gene overexpression plasmid vector on inhibiting cancer cells
  • Construction of human RNF20 gene overexpression plasmid vector and effect of human RNF20 gene overexpression plasmid vector on inhibiting cancer cells

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Embodiment 1: Preparation of human RNF20 gene overexpression plasmid vector

[0026] 1. Preparation of human RNF20 gene

[0027] Select the coding region sequence of human RNF20 (NM_019592.7) gene from Genbank of NCBI website, use Primer5 software, according to the pcMV-Tag2B plasmid vector map, combined with the existing restriction endonucleases in our laboratory, select BamHI and HindIII two Restriction site, design the upstream primer and downstream primer of human RNF20 gene. Using the cDNA of human mesenchymal stem cell HMSC as a template, the sequence was amplified by PCR technology to obtain the target gene, namely human RNF20 gene. Wherein, the upstream primer sequence of the human RNF20 gene designed by Primer5 software is SEQ ID NO.1, and the downstream primer sequence is SEQ ID NO.2. The reaction system for sequence amplification using PCR technology is shown in Table 1. PCR technology is used to amplify the sequence. The reaction conditions of the increas...

Embodiment 2

[0041] Example 2: Using real-time fluorescent quantitative RT-PCR to detect the high expression efficiency of human RNF20 gene after transfection of pcMV-Tag2B-RNF20 plasmid vector

[0042]Human HT29 cells in logarithmic growth phase were digested with trypsin, prepared into cell suspension with D-MEM / F-12 medium containing 10% AusGeneX serum, and 1.2×10 6 cells were seeded in a 6-well plate, which was then placed at 37 °C, 5% CO 2 cultured in an incubator to allow the cells to adhere to the wall. Transfection can be started when the cell density grows to 70-80%, and the transfection mixture is prepared according to the transfection system according to the plasmid concentration and the instructions of the transfection reagent TurboFect. Among them, the 6-well plate transfection system is shown in Table 5.

[0043] Table 5: 6-well plate transfection system

[0044]

[0045] Place the previous transfection mixture at 37 °C, 5% CO 2 Incubate in an incubator for 20 minutes,...

Embodiment 3

[0052] Example 3: Detection of proliferation ability of cancer cells transfected with pcMV-Tag2B-RNF20 plasmid

[0053] Human HCT116 and HT29 cells in the logarithmic growth phase were digested with trypsin, and prepared into cells with D-MEM / F-12 medium containing 10% AusGeneX serum and DMEM high-glucose medium containing 10% AusGeneX serum, respectively Suspension, 4~5×10 per well 4 cells were seeded in a 96-well plate, and then placed at 37 °C, 5% CO 2 cultured in an incubator to allow the cells to adhere to the wall. Transfection can be started when the cell density grows to 70-80%, and the transfection mixture is prepared according to the transfection system according to the plasmid concentration and the instructions of the transfection reagent TurboFect. Among them, the 96-well plate transfection system is shown in Table 8.

[0054] Table 8: 96-well plate transfection system

[0055]

[0056] Place the previous transfection mixture at 37 °C, 5% CO 2 Incubate in t...

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Abstract

The invention relates to construction of a human RNF20 gene overexpression plasmid vector and an effect of the human RNF20 gene overexpression plasmid vector on inhibiting cancer cells. The inventiondiscloses the effect of the human RNF20 gene on inhibiting colon cancer cells through in-vitro cell experimental study on the human RNF20 gene overexpression vector. The human RNF20 gene overexpression vector constructed by the invention can efficiently infect target cells, so that proliferation and migration of the colon cancer cells are inhibited, apoptosis of the colon cancer cells is promoted,and the human RNF20 gene overexpression vector has important significance in treatment of colon cancer and research and development of related anti-cancer drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a construction process of a human RNF20 gene overexpression plasmid vector and its effect on inhibiting cancer cells. Background technique [0002] In yeast and mammals, ubiquitin is a highly conserved biomacromolecule containing 76 amino acid polypeptide chains with a molecular weight of 8.5kDa. Histone ubiquitination is an energy-driven enzyme-linked reaction process that requires the sequential activation of three types of enzymes: ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. At present, the well-studied is histone H2B monoubiquitination (H2Bub1). H2Bub1 is regulated by E3 ubiquitin ligase Bre1 in yeast, and occurs on the 123th lysine of histone H2B. In mammals, it occurs at Lysine 120 of histone H2B is mainly regulated by the heterodimer complex RNF20 / RNF40. [0003] RNF20 (ring fingerprotein 20) is a ring finger protein c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N15/52
CPCC12N15/85C12N15/66C12N9/93C12Y603/02019C12N2800/107
Inventor 周飒马文建蔡玉巧赵军刘心怡薛佳敏
Owner TIANJIN UNIV OF SCI & TECH
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