Construction of ADAMTS13-pEGFP-N1 vector and its expression in HeLa cells.
10.7534/j.issn.1009-2137.2013.01.026
- Author:
Jing LING
1
;
Zhen-Ni MA
;
Jian SU
;
Chang-Geng RUAN
Author Information
1. Jiangsu Institute of Hematology, Jiangsu Province, China.
- Publication Type:Journal Article
- MeSH:
ADAM Proteins;
genetics;
ADAMTS13 Protein;
Gene Expression;
Genetic Vectors;
Green Fluorescent Proteins;
genetics;
HeLa Cells;
Humans;
Transfection
- From:
Journal of Experimental Hematology
2013;21(1):126-129
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed to construct a pEGFP-N1 vector of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thrombospondin type 1 motifs 13) so as to pave the way for further studying its synthesis and secretion. Human full-length cDNA sequence of ADAMTS13 was acquired by polymerase chain reaction (PCR) with Phusion(®) High-Fidelity (NEB), then the PCR product was double digested with EcoRI and XhoI. After digestion, the ADAMTS13 cDNA sequence was purified and recombined with the pEGFP-N1 vector. The DNA sequence analysis showed that ADAMTS13 was ligated to the pEGFP-N1 vector correctly. After transient expression in HeLa cells, the expression of EGFP could be detected by fluorescent microscopy, and the expression of ADAMTS13 protein could be detected by SDS-PAGE and Western blot. It is concluded that the ADAMTS13-pEGFP-N1 vector is successfully constructed, and it can be widely used in further research on the mechanism of the synthesis and secretion of ADAMTS13.
