Construction and expression of dendritic cell nucleic acid vaccine containing CML28 gene in human dendritic cells.
- Author:
Dong-Hua ZHANG
1
;
Hong-Sheng ZHOU
;
Ya-Ya WANG
;
Wen-Li LIU
;
Zhen-Qian HUANG
;
Huo TAN
Author Information
1. Hematopoietic Stem Cell Transplantation Department, Tongji Organ Transplantation Research Institute, China. zdh62@yahoo.com.cn
- Publication Type:Journal Article
- MeSH:
Antigens, Neoplasm;
genetics;
immunology;
metabolism;
Antigens, Surface;
genetics;
immunology;
metabolism;
Blotting, Western;
Cells, Cultured;
Cloning, Molecular;
DNA, Complementary;
genetics;
Dendritic Cells;
cytology;
immunology;
metabolism;
Electrophoresis, Polyacrylamide Gel;
Exoribonucleases;
genetics;
immunology;
metabolism;
Exosome Multienzyme Ribonuclease Complex;
Flow Cytometry;
Genetic Vectors;
genetics;
Humans;
K562 Cells;
RNA-Binding Proteins;
Recombinant Fusion Proteins;
biosynthesis;
genetics;
immunology;
Transfection;
methods;
Vaccines, DNA;
biosynthesis;
genetics;
immunology
- From:
Journal of Experimental Hematology
2005;13(4):631-636
- CountryChina
- Language:English
-
Abstract:
This study was aimed to construct nucleic acid vaccine containing the coding region of the CML28 gene and to express it in human dendritic cells. The full length of CML28 cDNA was amplified from K562 by RT-PCR and subcloned into pGEM-T vector. The CML28 fragment was digested and subsequently inserted into the EcoRI-Xba I sites of pcDNA3.1HisA to construct the recombinant expression vector pcDNA3.1HisA-CML28, which was identified by restrition analysis and sequencing. Human dendritic cells (DC) were separated from peripheral blood mononuclear cells (PBMC) by culture with rhGM-CSF, rhIL-4 and assessed by flow cytometry. The constructed plasmid pcDNA3.1 HisA-CML28 was transfected into DC by electroporation. Western blot was used to detect the expression of fusion protein His-CML28. The results showed that recombinant plasmid pcDNA3.1HisA-CML28 contained the correct full CML28 cDNA identified by restriction analysis and sequencing, and can express the fusion protein His-CML28 in DCs. It is concluded that nucleic acid vaccine containing CML28 gene was constructed and expressed in DC successfully.
