title: [Construction of ADAMTS13-pEGFP-N1 vector and its expression in HeLa cells].
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.
authors.affiliation: Jiangsu Institute of Hematology, Jiangsu Province, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Ling
authors.forename: Zhen-Ni
authors.initials: ZN
authors.lastname: Ma
authors.forename: Jian
authors.initials: J
authors.lastname: Su
authors.forename: Chang-Geng
authors.initials: CG
authors.lastname: Ruan
mesh_terms: D051722:ADAM Proteins / Q000235:genetics*; D000071120:ADAMTS13 Protein; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D014162:Transfection
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human