Construction of ADAMTS13-pEGFP-N1 vector and its expression in HeLa cells / 中国实验血液学杂志
Journal of Experimental Hematology
; (6): 126-129, 2013.
Article
em Zh
| WPRIM
| ID: wpr-325199
Biblioteca responsável:
WPRO
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.
Texto completo:
1
Índice:
WPRIM
Assunto principal:
Células HeLa
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Transfecção
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Expressão Gênica
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Proteínas de Fluorescência Verde
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Proteínas ADAM
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Proteína ADAMTS13
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Vetores Genéticos
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Genética
Limite:
Humans
Idioma:
Zh
Revista:
Journal of Experimental Hematology
Ano de publicação:
2013
Tipo de documento:
Article