Construction of eukaryotic expressing vector of multiple myeloma mucin-1 and its expression in COS-7 cells in vitro.
- Author:
Kun LIU
1
;
Yun-Jiao LUO
;
Yue-Bo LIU
;
Jin YAO
;
Hong YANG
;
Hong MOU
;
Gui-Yun HUANG
;
You ZHANG
Author Information
1. Department of Hematology, The Second Hospital of Kunming Medical University, Kunming 650101, Yunnan Province, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Base Sequence;
COS Cells;
Cercopithecus aethiops;
Genetic Vectors;
Molecular Sequence Data;
Mucin-1;
genetics;
Multiple Myeloma;
genetics;
Plasmids;
Transfection
- From:
Journal of Experimental Hematology
2009;17(4):898-902
- CountryChina
- Language:Chinese
-
Abstract:
In order to construct an eukaryotic expression vector for gene of multiple myeloma mucin1 (muc1-2vntr) gene and to express it in COS-7 cells in vitro, so to provide the basic material for further research of multiple myeloma DNA vaccine. muc1-2vntr coding gene was used as a research gene and a KOZAK sequence was inserted before the gene Hind III and XbaI restriction sites were inserted before and after the coding gene. Then the whole sequence was synthesized and inserted into pcDNA3.1/myc-his B vector, and the resulted recombinant vector was transformed into E.coil competent cells to get an engineering strain, the recombinant plasmid pcDNA3.1-2vntr/myc-his B identified by restriction analysis and DNA sequencing were transfected into COS-7 cells by liposome-mediated gene transfer method. Finally, fluorescent microscopy was used to assess GFP expression and Western blot analysis using muc1 monoclonal antibody was used to recognize vntr, confirming the expression of vntr. The results showed that the full length of synthesized muc1-2vntr gene, as expected, was 140 bp. Both restriction analysis and DNA sequencing demonstrated that pcDNA3.1-2vntr/myc-his B included the whole translation frame region and muc1-2vntr gene. Furthermore, the fluorescence microscopy proved that the recombinant plasmid had been successfully transfected into COS-7 cells. The expression of mucin-1 protein was observed both in the transfected cell and the cell supernatant by Western blot. It is concluded that the pcDNA3.1-2vntr/myc-his B has been successfully constructed and expressed in COS-7 cells in vitro, which provides the basic material for further researches of mucin-1 function and possible multiple myloma DNA vaccine.
