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Application of human TMEFF1 gene and related products

A kind of gene and use technology, applied in the use of human TMEFF1 gene and related product fields

Active Publication Date: 2020-03-24
BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no relevant report on the use of TMEFF1 gene in the treatment of leukemia

Method used

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  • Application of human TMEFF1 gene and related products
  • Application of human TMEFF1 gene and related products
  • Application of human TMEFF1 gene and related products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1 Preparation of RNAi lentivirus against human TMEFF1 gene

[0108] 1. Screening for effective siRNA targets against human TMEFF1 gene

[0109] Retrieve TMEFF1 (NM_003692) gene information from Genbank; design effective siRNA targets for TMEFF1 gene. Table 1-1 lists the screened effective siRNA target sequences against the TMEFF1 gene.

[0110] Table 1-1 is targeted at the siRNA target sequence of human TMEFF1 gene

[0111] SEQ ID NO TargetSeq(5'-3') 1 TGCCAATTTCAGTGCCATA

[0112] 2. Preparation of lentiviral vector

[0113] 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.

[0114] Table 1-2 Double-stranded DNA Oligo with sticky end...

Embodiment 2

[0133] Example 2 Detection of gene silencing efficiency of tumor cells infected with TMEFF1-siRNA lentivirus

[0134] Human leukemia cells Jurkat and human leukemia cells MOLT-4 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, Jurkat: 25, MOLT-4: 150), an appropriate amount of the lentivirus prepared in Example 1 was added, the culture medium was replaced after 24 hours of cultivation, and the cells were collected after the infection time reached 5 days.

[0135] a) Detection of gene silencing efficiency by real-time fluorescent quantitative RT-PCR

[0136] 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...

Embodiment 3

[0172] Example 3 Detection of proliferation ability of tumor cells infected with TMEFF1-siRNA lentivirus

[0173]Human leukemia Jurkat cells and human leukemia MOLT-4 cells in the logarithmic growth phase were trypsinized to make cell suspension (the number of cells was about 5×104 / ml) and inoculated in 6-well plates, and cultivated to the degree of cell confluence to about 30%. According to the multiplicity of infection (MOI, Jurkat: 25, MOLT-4: 150), 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 complete the medium Resuspend into a cell suspension and count. Determine the cell density (3000cell / 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 group, fine-tune the amount of c...

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Abstract

The invention,, which belongs to the field of biomedical research, particularly relates to application of a human TMEFF1 gene as a target in preparation of leukemia treatment drugs or leukemia diagnosis drugs. The wide and deep research finds that proliferation of leukemia cells can be effectively inhibited and apoptosis can be promoted after expression of the human TMEFF1 gene is down-regulated by adopting an RNAi method and thus the growth process of leukemia tumor cells can be effectively controlled. The siRNA or the nucleic acid construct or lentivirus containing the siRNA sequence can specifically inhibit the proliferation rate of leukemia tumor cells, promote apoptosis of the leukemia tumor cells, inhibit cloning of the leukemia tumor cells, influence the period of the leukemia tumorcells and inhibit development of leukemia, so that the leukemia is treated. Therefore, an novel direction is opened up for leukemia treatment.

Description

technical field [0001] The invention belongs to the field of biomedical research, and specifically relates to the use of human TMEFF1 gene and related products. Background technique [0002] TMEFF1 is a protein-coding gene. TMEFF1 is a member of the CTA family (CT 120-1) (Condomines M, Hose D, Rème T, et al. Gene expression profiling and real-time PCR analysesidentify novel potential cancer-testis antigens in multiple myeloma. J Immunol. 2009; 183 (2) :832–840.). As a transmembrane protein, TMEFF1 is highly expressed in human embryonic and neural tissues. Early studies mainly focused on the involvement of TMEFF1 in the middle and late embryonic development and its regulation on the central nervous system (EibDW, Martens GJ. A novel transmembrane protein with epidermal growth factor and follistatin domains expressed in the hypothalamo-hypophysial axis of Xenopuslaevis. J Neurochem.1996; 67(3):1047–1055.). [0003] Leukemia is a malignant clonal disease of hematopoietic st...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867A61K45/00A61K31/713A61P35/00C12Q1/6886
CPCC12N15/113C12N15/867A61K45/00A61K31/713A61P35/00C12Q1/6886C12Q2600/136C12Q2600/158C12N2740/15043C12N2800/107C12N2310/14C12N2310/531
Inventor 高超郑胡镛
Owner BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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