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ANGPTL4 deletion mutant and application thereof

A technology for deletion of mutants and mutants, applied in the field of new tumor-related genes, can solve the problem of not knowing or understanding deletions or fragments, and achieve broad application value and the effect of promoting tumor angiogenesis

Inactive Publication Date: 2007-04-25
SHANGHAI INST OF ONCOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the research on the full-length gene sequence and its function is relatively common, but people do not know or understand its deletion or fragment

Method used

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  • ANGPTL4 deletion mutant and application thereof
  • ANGPTL4 deletion mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Construction of ANGPTL4 deletion mutants

[0099] The results of bioinformatics analysis of ANGPTL4 showed that the ANGPTL4 gene was located on chromosome 19p13.3, containing 7 exons and 6 introns; the full length of the gene cDNA was 1943bp, the open reading frame contained 1218bp, encoding 406 amino acids , with an estimated molecular weight of 45.2 kDa. The N-terminus has a hydrophobic signal peptide and coiled-coil (Coiled-Coil) domain, and the C-terminus has a fibrinogen-like (Fibrinogen-like) domain (Figure 1A).

[0100] In this example, the mammalian cell expression vector EGFP-N1 (purchased from Clontech Company) was used to construct the full-length ANGPTL4 gene and three deletion mutant subclones (Fig. 1B). Sexual primers were used to amplify the coding region of the ANGPTL4 gene (1-406 amino acids) and its three ANGPTL4 deletions Δ1 (1-191 amino acids), Δ2 (67-406 amino acids), Δ3 (183- 406 amino acids), were loaded into EGFP-N1 vectors with XhoI / KpnI restr...

Embodiment 2

[0102] Cellular localization of ANGPTL4 protein deletion mutant fusion protein in human hepatoma cell SMMC-7721

[0103]It can be seen from Figure 2 that the EGFP protein is evenly distributed in the nucleus, cytoplasm and membrane; the full-length gene of ANGPTL4 is localized in the cytoplasm, and the deletion mutant (pp1158-delta1) lacking the C-terminal Fibronectin functional domain and only missing the N-terminal signal peptide The overexpression product of the deletion mutant (pp1158-delta2) is mainly localized in the nucleus and distributed in a punctate manner. In the deletion mutant (pp1158-delta 3) lacking the N-terminal signal peptide and Coiled-coil functional domain, the overexpression product was mainly localized in the nucleus, but evenly distributed.

Embodiment 3

[0105] Establishment of ANGPTL4 gene and deletion mutant stable cell lines and detection by Western blot

[0106] LipofectAMINE liposomes were used to transfect EGFP-N1 empty vector, ANGPTL4-GFP-N1 (full length), ANGPTL4-delta1-GFP-N1, ANGPTL4-delta2-GFP-N1, ANGPTL4-delta3-GFP-N1 fusion expression vector, See Figure 3 for the Western blot detection of the liver cancer cell line SMMC7721 cells screened by G418.

[0107] The results showed that all the target genes were expressed, indicating that the stable cell line was successfully established.

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Abstract

The deletion mutant of gene ANGPTL4 and its application relates to a new cancer-related gene, particularly to the function application of gene ANGPTL4. ANGPTL4-Delt1, the deletion mutant of gene ANGPTL4, losses C-terminal functional domain of fibronectin; deletion mutant ANGPTL4-Delt2 losses the N-terminal signal peptide; deletion mutant ANGPTL4-Delt3 losses the N-terminal signal peptide and curly functional domain. The deletion mutant of gene ANGPTL4 has the effect of inhibiting the growth of liver cancer cells in vitro, and can be used to produce drugs for cancer inhabiting.

Description

technical field [0001] The invention relates to new tumor-related genes, in particular to ANGPTL4 deletion mutants and applications thereof. Background technique [0002] With the completion of the Human Genome Project and the continuous advancement of science and technology, the "post-genome era" with the main content of research on the function of genes (especially disease-causing genes) has come. [0003] Although genes related to various functions have been cloned successively at home and abroad, and their preliminary biological functions and mechanisms of action have been studied, the large-scale functional screening based on cell growth in our laboratory has discovered and cloned 372 genes. Novel genes [Wan et al., PNAS (2004) 101(44): 15724-15729]. [0004] The ANGPTL4 gene (originally named pp1158 by Li Jinjun's laboratory) [Zhu Hongxin et al., Chinese Journal of Oncology (2002) 24 (2): 123-125] was cloned from the human placenta cDNA library by using this functiona...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435A61K48/00A61P35/00C12N15/73C12P21/02
Inventor 李锦军张锋锐顾建人
Owner SHANGHAI INST OF ONCOLOGY
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