Application of human DNAJC24 gene and related products

A gene and application technology, applied in the field of application of human DNAJC24 gene and related products, can solve problems such as the absence of DNAJC24 gene

Inactive Publication Date: 2020-04-28
TIANJIN MEDICAL UNIV CANCER INST & HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, there is no relevant report on th

Method used

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  • Application of human DNAJC24 gene and related products
  • Application of human DNAJC24 gene and related products
  • Application of human DNAJC24 gene and related products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1 Preparation of RNAi lentivirus against human DNAJC24 gene

[0115] 1. Screening for effective siRNA targets against human DNA JC24 gene

[0116] Retrieve DNAJC24 (NM_181706) gene information from Genbank; design effective siRNA targets for DNAJC24 gene. Table 1-1 lists the screened effective siRNA target sequences against the DNAJC24 gene.

[0117] Table 1-1 is targeted at the siRNA target sequence of human DNAJC24 gene

[0118] SEQ ID NO TargetSeq(5'-3') 1 AAGTACAGATGTACCAGCA

[0119] 2. Preparation of lentiviral vector

[0120] 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.

[0121] Table 1-2 Double-stranded DNA Oligo with sti...

Embodiment 2

[0139] Example 2 Detection of Silencing Efficiency of Tumor Cells Infected with DNAJC24-siRNA Lentivirus

[0140] The human lung cancer A549 and NCI-H1299 cells in the logarithmic growth phase were trypsinized 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, A549:10, NCI-H1299:10), 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.

[0141] a) Detection of gene silencing efficiency by real-time fluorescent quantitative RT-PCR

[0142] 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 r...

Embodiment 3

[0177] Example 3 Detection of proliferation ability of tumor cells infected with DNAJC24-siRNA lentivirus

[0178] a) Celigo experiment

[0179]The human lung cancer A549 and NCI-H1299 cells in the logarithmic growth phase were trypsinized 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, A549: 10, NCI-H1299: 10), add an appropriate amount of virus, replace the medium after 24 hours of culture, and collect cells in each experimental group in the logarithmic growth phase after the infection time reaches 5 days . The complete medium was resuspended into a cell suspension (2×10 4 / ml), at a cell density of about 1500 / well (A549), 1200 / well (NCI-H1299), inoculate a 96-well plate. 5 replicate wells in each group, 100 μl per well. After laying the board, place at 37°C, 5% CO 2 Incubator cultivation. From the second d...

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Abstract

The invention belongs to the field of research of biomedicines, and particularly relates to application of a human DNAJC24 gene as a target to preparation of medicines for treating lung cancers or medicines for diagnosing the lung cancers. Based on extensive and profound researching finding, by adopting a RNAi method, expression of the human DNAJC24 is regulated-down, so that proliferation of thelung cancer cells can be effectively inhibited and cell apoptosis is promoted, and a growth progress of the lung cancers can be effectively controlled. According to the siRNA or a nucleic acid construction body including an siRNA sequence and chronic viruses provided by the invention, the proliferation velocity of the lung cancer cells can be specifically inhibited, the cell apoptosis of the lungcancers is promoted, cell cloning of the lung cancers is inhibited, cell invasion of the lung cancers is inhibited, cell transferring of the lung cancers is inhibited, and growth of the lung cancers is inhibited, so that the lung cancers can be treated, and a new direction is developed for treatment of the lung cancers.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the application of human DNA JC24 gene and related products. Background technique [0002] Diphtheria amide is a unique post-translational modification of histidine found only in translation elongation factor-2 (EEF2), which is well conserved from archaea to humans, and is responsible for diphtheria toxin (DT) and exotoxin A A target for ADP-ribosylation and inactivation of EEF2, and DNAJC24 is one of the enzymes involved in diphtheria acylation of EEF2. The disease associated with DNAJC24 is Aniridia 1, and its associated pathways include γ-carboxylation of proteins, formation of hydroxyproline, and activation and metabolism of arylsulfatases. Studies have found that many patients with acute lymphoblastic leukemia are resistant to the immunotoxin HA22. In a HA22-resistant ALL cell line, it was found that the expression level of the DANJC24 gene promoter was signifi...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/713A61K31/7105A61K39/395A61K48/00A61P35/00A61P35/04C12N15/113C12N15/867C12N7/01C12Q1/6886
CPCA61K31/7105A61K31/713A61K39/39558A61K45/00A61K48/005A61P35/00A61P35/04C12N7/00C12N15/113C12N15/86C12N2310/14C12N2740/15021C12N2740/15043C12Q1/6886C12Q2600/158
Inventor 郭华陈鹏刘志勇林丽陈璐陈丽维罗艺刘东明张翠翠
Owner TIANJIN MEDICAL UNIV CANCER INST & HOSPITAL
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