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Use of sorting protein 33 in treatment of tumor
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A tumor cell and gene technology, applied in microorganism-based methods, microbial assay/test, microorganisms, etc., can solve the problem that the ability of β-secretase to split has no effect, etc.
Active Publication Date: 2009-12-02
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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Exogenous expression of sortin 33 in the culture system can increase the ability of APPα-secretase to split, but has no effect on the ability of β-secretase to split. This phenotype is similar to the result of Dynamin1-inhibiting mutant K44A (Schobel S, Neumann S , Hertweck M et al., J Biol Chem.2008, 283: 14257-14268)
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[0042] Human cell lines (A549, Pro5, PC3, SPCA1, NCIH460, HT1080, Hlamp, HEK293T, HT1080, GLC82, L-78, MCF7 and Hela) were cultured in medium supplemented with 10% fetal bovine serum FBS (Hyclone) at 37 Cultivate in an incubator at ℃ (see Qin B, He M, Chen X et al., J Biol Chem. 2006, 281: 36891-36896).
Embodiment 2
[0043] Example 2: Expression of SNX33 protein (SEQ ID NO: 2) in different cell lines
[0044] First, different human cell lines (A549, Pro5, PC3, SPCA1, NCIH460, HT1080, Hlamp, HEK293T, HT1080, GLC82, L-78, MCF7, and Hela) were lysed with RIPA lysate to obtain protein lysate. Such as figure 1As shown, by Western-Blotting analysis, we found that in the 13 cell lines of A549, Pro5, PC3, SPCA1, NCIH460, HT1080, Hlamp, HEK293T, HT1080, GLC82, L-78, MCF7 and Hela, except for the Hlamp cell line In addition to less expression, SNX33 is highly expressed in most cells. Among them, GAPDH is an internal reference, which shows that the amount of protein we use is basically the same. After the SNX33 protein was blocked with the SNX33 antibody overnight, the analyzed cell line was detected again, and the specific band of SNX33 disappeared, indicating that the target band detected by the SNX33 antibody was the SNX33 protein. . This example demonstrates that sortin 33 is ubiquitously expr...
Embodiment 3
[0045] Embodiment 3: Construction of SNX33 vector (SEQ ID NO: 1)
[0046] 2ug of total RNA was reverse transcribed using a reaction system with a final volume of 20ul (see Pan, G et al., (2006) Faseb J 20(10), 1730-1732) to obtain a cDNA library. The human SNX33 fragment was amplified using the primers in Table 1, and then recovered by agarosegel electrophoresis.
[0047] Table 1: Primers for cloning SNX33 fragments:
[0048] SEQ ID NO
[0049] The plasmid for eukaryotic expression was constructed by inserting PCR fragments into the multiple cloning site EcoR V of the pCR3.1 vector fused with green fluorescent protein GFP, that is, the pCR3.1 vector was digested by EcoR V for 4 hours at 37 ° C, Then, the human SNX33 fragment amplified from the cDNA of human embryonic kidney cell HEK293T was ligated in the presence of ligase at 16° C. for 4 hours to obtain the eukaryotic expression vector GFP-SNX33.
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Abstract
The invention discloses use of human sorting protein 33 in the treatment of tumor. As a member of the sorting protein family, the human sorting protein 33 can be widely expressed in tumor cells. Different from other sorting protein, the human sorting protein 33, apart from roles played in a process of protein endocytosis and transfer, is proved to have a significant effect on the growth of the tumor cells that over expression or inhibition of the sorting protein 33 in the tumor cells result in the stop of the growth and consequent death of the tumor cells. The human sorting protein 33 lays a thoetherical foundation for the sorting protein 33-targetted design and development of antitumor medicaments. In the invention, the influences of the over expression and the inhibition of the sorting protein 33 on the growth of the tumor cells are explained mechanically, so the sorting protein 33 is proved to have an effect of inhibiting the growth of the tumor cells and an application mode of the storing protein 33 gene as a new means or medicament for treating tumor is provided.
Description
Technical field: [0001] The present invention relates to the construction of an expression vector of sorting protein 33 (Sorting Nexin 33) and its use in tumor therapy. The sorting protein 33 is a member of the sorting protein family. Functional studies in tumor cells are very little. Background technique: [0002] One of the main differences between eukaryotic cells and prokaryotic cells is the presence of a variety of membranous organelles, such as the nucleus, Golgi complex, endoplasmic reticulum, lysosomes, mitochondria, and chloroplasts in plants. In addition, another class of organelles with no obvious characteristics exists in the cytoplasm, which regulate the transport and communication of important substances between the inside and outside of the cell. Endosomes are a kind of vesicleassembly, which are composed of various substances swallowed by the plasma membrane of the cell, including cell surface receptor proteins and their binding ligand proteins, nutritional...
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IPC IPC(8): C12Q1/68C12Q1/02C12R1/91
Inventor 裴端卿张娟张小飞
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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