Application of human GRPEL2 gene and relevant product

A gene and application technology, applied in medical preparations containing active ingredients, genetic engineering, plant genetic improvement, etc.

Pending Publication Date: 2020-06-19
宋程
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Application of human GRPEL2 gene and relevant product
  • Application of human GRPEL2 gene and relevant product
  • Application of human GRPEL2 gene and relevant product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Example 1 Preparation of RNAi lentivirus against human GRPEL2 gene

[0107] 1. Screening for effective siRNA targets against the human GRPEL2 gene

[0108] Retrieve GRPEL2 (NM_152407) gene information from Genbank; design effective siRNA targets for GRPEL2 gene. Table 1-1 lists the screened effective siRNA target sequences against the GRPEL2 gene.

[0109] Table 1-1 is targeted at the siRNA target sequence of human GRPEL2 gene

[0110] SEQ ID NO TargetSeq(5'-3') 1 GGCTCTATTTGGGTAATTT

[0111] 2. Preparation of lentiviral vector

[0112] 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.

[0113] Table 1-2 Double-stranded DNA Oligo with sticky...

Embodiment 2

[0131] Example 2 Real-time fluorescent quantitative RT-PCR method to detect gene silencing efficiency

[0132] Human colorectal cancer HCT116 cells and colorectal cancer RKO 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, HCT116:10, RKO:10), an appropriate amount of the lentivirus prepared in Example 1 was added, the culture medium was replaced after 24 hours of culture, and the cells were collected after the infection time reached 5 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 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...

Embodiment 3

[0139] Example 3 Detection of proliferation ability of tumor cells infected with GRPEL2-siRNA lentivirus (MTT experiment)

[0140] Human colorectal cancer HCT116 cells and colorectal cancer RKO 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, HCT116:10, RKO:10), add an appropriate amount of virus, replace the medium after 24 hours of culture, collect cells in each experimental group in the logarithmic growth phase, trypsinize, and resuspend in complete medium into a cell suspension and counted. Determine the cell density (2500cell / well) according to the growth rate of the cells. Repeat 3-5 times in each group. After the cells are completely settled, observe the cell density of each experimental group under a microscope. If the density is uneven, Fix one...

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Abstract

The invention belongs to the field of biomedical research, and particularly relates to an application of a human GRPEL2 gene as a target to preparation of a drug for treating colorectal cancer. According to extensive and in-depth research, after expression of the human GRPEL2 gene is down-regulated by a RNAi method, proliferation of colorectal cancer cells can be effectively inhibited, apoptosis can be effectively promoted, and the growth process of colorectal cancer can be effectively controlled. The provided siRNA, or a nucleic acid construct and lentivirus containing the siRNA sequence canspecifically inhibit the proliferation rate, promote apoptosis and inhibit cloning, invasion and metastasis of the colorectal cancer cells, thereby treating the colorectal cancer and opening up a newdirection for colorectal cancer treatment.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human GRPEL2 gene and related products. Background technique [0002] GRPEL2 (GrpE Like 2, Mitochondrial) is located on chromosome 5q32 and is a protein-coding gene. The encoded protein is mainly located in the mitochondria, followed by the cytoplasm. The PAM complex is a complex that transports the constituent proteins of the transit peptide from the mitochondrial membrane to the matrix in an ATP-dependent manner. GRPEL2 is the main component of the PAM complex and forms a homodimer with GRPEL1. Under oxidative stress conditions, the formation of GRPELs dimers can promote the entry of proteins related to the folding mechanism into the mitochondrial matrix, or enhance the chaperone activity of mitochondrial HSP70, thereby protecting the protein stability of mitochondria under oxidative stress. As a class of nucleotide exchange factor homologues, GrpEL1 a...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/867C12N15/113A61K45/00A61P35/00
CPCC12Q1/6886C12N15/86C12N15/113A61K45/00A61P35/00C12N2740/15043C12N2310/141
Inventor 宋程唐蔚潘博候公瑾
Owner 宋程
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