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Application of human HIST1H2BK gene and related products

A technology of HIST1H2BK and gene, which is applied in the field of application of human HIST1H2BK gene and related products, and can solve the problem of no literature report on the function of HIST1H2BK

Pending Publication Date: 2020-03-24
广西医科大学附属肿瘤医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no literature reporting the role of HIST1H2BK in the occurrence and development of liver cancer

Method used

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  • Application of human HIST1H2BK gene and related products
  • Application of human HIST1H2BK gene and related products
  • Application of human HIST1H2BK gene and related products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 Preparation of RNAi lentivirus against human HIST1H2BK gene

[0101] 1. Screening for effective siRNA targets against the human HIST1H2BK gene

[0102] Retrieve HIST1H2BK (NM_080593) gene information from Genbank; design effective siRNA targets for HIST1H2BK gene. Table 1-1 lists the screened effective siRNA target sequences against the HIST1H2BK gene.

[0103] Table 1-1 is targeted at the siRNA target sequence of human HIST1H2BK gene

[0104] SEQ ID NO TargetSeq(5'-3') 1 GAGAAAGAGTATATAAGCT

[0105] 2. Preparation of lentiviral vector

[0106] Aim at the siRNA target (take SEQ ID NO: 1 as an example) to synthesize a double-stranded DNA Oligo sequence (Table 1-2) with Age I and EcoR I restriction sites at both ends; Dicer acts on the pGCSIL-GFP vector (provided by Shanghai Jikai Gene Chemical Technology Co., Ltd.) to linearize it, and agarose gel electrophoresis identifies the digested fragment.

[0107] Table 1-2 Double-stranded DNA...

Embodiment 2

[0125] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency

[0126] 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), an appropriate amount of the lentivirus prepared in Example 1 was added, and the culture medium was replaced after 24 hours of culture. After the infection time reached 5 days, the cells were collected. 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 2-1 for the reverse transcription reaction system, react at 42°C for 1 hour, and then bathe in a water bath at 70°C for 10 minutes to inactivate the reverse ...

Embodiment 3

[0133] Example 3 Western Blotting method to detect gene silencing efficiency

[0134] 1. Extraction of total cell protein

[0135] 1) Infect the target cells (SMMC-7721 cells) with the control virus and the RNAi virus targeting the HIST1H2BK interference target according to the multiplicity of infection (MOI: 10).

[0136] 2) After 5 days of infection, collect the cell samples, take an appropriate amount of RIPA lysate (Beiyuntian, P0013C), and add PMSF within a few minutes before use, so that the final concentration of PMSF is 1mM.

[0137] 3) Add an appropriate amount of RIPA lysate, and lyse on ice for 10-15 minutes. Scrape the cells and transfer them into a new EP tube, and then ultrasonically disrupt the cells (20 times at 40W, 1 s each time, 2 s apart).

[0138] 4) Centrifuge at 12000g for 15min at 4°C, take the supernatant and use BCA Protein Assay Kit (manufacturer: Biyuntian, article number: P0010S) to measure the protein concentration.

[0139] 5) Add new lysate t...

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Abstract

The invention belongs to the field of biomedical research, and particularly relates to application of human HIST1H2BK gene as a target in preparation of drugs used for treating liver cancer. It is found through wide and deep research that proliferation of liver cancer cells can be effectively inhibited, apoptosis can be promoted and the growth process of liver cancer can be effectively controlledafter the expression of human HIST1H2BK gene is down-regulated by adopting an RNAi method. A siRNA provided by the invention, or a nucleic acid construct and lentivirus containing the siRNA sequence can specifically inhibit the proliferation rate of liver cancer cells, promote apoptosis of liver cancer cells, inhibit cloning of liver cancer cells and inhibit growth of liver cancer cells, so that liver cancer is treated, and a new direction is provided for liver cancer treatment.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human HIST1H2BK gene and related products. Background technique [0002] Histones are the basic nuclear proteins responsible for the structure of the nucleosome of chromosome fibers in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer around which approximately 146 bp of DNA is wrapped, and this repeating unit is called a nucleosome . Linker histone H1 interacts with linker DNA between nucleosomes and plays a role in the formation of high-level structures in chromatin. The protein encoded by the HIST1H2BK gene is a replication-dependent histone, one of the members of the histone H2B family. It is the core component of the nucleosome and has the activity of resisting bacteria and fungi. Currently, the HIST1H2BK gene contains two transcripts encoding the same protein, located on chromosome 6p21.33, and expr...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867A61K45/00A61K31/713A61P35/00C12Q1/6886
CPCC12N15/113C12N15/867A61K45/00A61K31/713A61P35/00C12Q1/6886C12Q2600/136C12Q2600/158C12N2310/14C12N2740/15043C12N2800/107C12N2310/531
Inventor 陈闯甘激文邬国斌黄山李科志何剑波王宗玉林栋毅
Owner 广西医科大学附属肿瘤医院
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