Cloning of human angiogenesis inhibitor METH1 and its expression in mammalian cells.
- Author:
Yang ZHANG
1
;
Kai-hua LU
;
Shu-zhong GUO
;
Ge SONG
;
Guo-rong YANG
;
Zhen WANG
;
Yong-hong LEI
Author Information
- Publication Type:Journal Article
- MeSH: ADAM Proteins; ADAMTS1 Protein; Angiogenesis Inhibitors; genetics; metabolism; Blotting, Western; Cell Line, Tumor; Cloning, Molecular; DNA, Complementary; genetics; metabolism; Disintegrins; genetics; metabolism; Humans; Metalloendopeptidases; genetics; metabolism; Reverse Transcriptase Polymerase Chain Reaction; Transfection; methods
- From: Chinese Journal of Plastic Surgery 2004;20(3):225-227
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo get the full length of human METH1 cDNA and express it steadily in mammalian cell stably.
METHODSMETH1 was amplified by RT-PCR, and cloned into pCDNA3.0 after confirmed by sequence analysis. HepG2 cells were transfected by Lipofectamine reagent and then selected in medium with G418. The expression level of METH1 was detected by RT-PCR and Western blot.
RESULTSMETH1 with expected length was effectively amplified, and completely matched the published sequence of encoding mature peptide [GI:5725505] as shown by sequence analysis. Eukaryotic vector expressing METH1 was obtained by gene cloning, cells expressing METH1 was got by selection with G418 at 3 weeks after transfection. RT-PCR and Western blot showed high level expression of METH1.
CONCLUSIONFull length of human METH1 gene is cloned successfully and expressed in HepG2 steadily, The results set up a basis for the study of effects of METH1 on hypertrophic scar angiogenesis.
