Construction and expression of eukaryotic expression vector harboring human neutrophil peptide 1
- VernacularTitle:人中性粒细胞多肽1真核表达载体的构建与表达
- Author:
Juan LIU
;
Yongtao SUN
;
Guangyu LI
- Publication Type:Journal Article
- Keywords:
human neutrophil peptide1 (HNP1);
gene clone;
eukaryotic expression vector
- From:
Medical Journal of Chinese People's Liberation Army
2001;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the possibility of human neutrophil peptide 1 (HNP1) gene engineering, we construct the eukaryotic expression vector carrying HNP1 gene. Methods With RNA extracted from human neutrophil cell as template, cDNA encoding mature HNP1 was amplified by RT-PCR, and then it was inserted into vector pMD18-T. After restriction endonuclease digestion and DNA sequencing confirmation the gene was subcloned into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO to construct a recombinant expression plasmid pcDNA3.1/V5-His-TOPO/HNP1, then the recombinant plasmid was transfected into COS-7 cells by lipofectamine, and the expressed product was identified by biotin-avidin enzyme-linked immunosorbent assay ( BA-ELISA). Results The sequence of HNP1 completely matched those published in GenBank, thus eukaryotic expression vector pcDNA3.1/V5-His-TOPO/HNP1 was constructed correctly. The ELISA results showed that the eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO/HNP1 could temporarily express HNP1 in COS-7 cells. Conclusion The successful construction and expression of pcDNA3.1/V5-His-TOPO/HNP1 pave the way for the stable expression HNP1 in mammalian engineering cells.
