[Cloning and expression of the extracellular region of human LIGHT gene in E.coli.].
OBJECTIVE
To construct a recombinant prokaryotic expression vector containing the extracellular region of human LIGHT gene and perform the express in E.coli.


METHODS
Total RNA was extracted from human immature bone marrow-derived dendritic cells. The extracellular region of human LIGHT gene was amplified by RT-PCR and cloned into pET32a(+) vector, the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing. After the recombinant plasmid was transformed into E.coli BL21 and induced with IPTG, the expressed protein was analyzed by SDS-PAGE and Western blot.


RESULTS
A 543 bp of the extracellular region of human LIGHT gene was obtained and the sequence was confirmed correct by DNA sequencing. SDS-PAGE and Western blot analysis showed that a protein with molecular weight of 41 000 was expressed in E.coli BL21.


CONCLUSIONS
The extracellular region of LIGHT gene is cloned successfully and expressed in E.coli BL21 and the elementary expression conditions were obtained, which lays a basis on the further functional research of LIGHT.
Hao
Wen-Li
WL
School of Life Science, East China Normal University, Shanghai 200062, China.
Jiang
Wen-Zheng
WZ
Wen
Jie-Jun
JJ
Fan
Yan
Y
Qian
Min
M
D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids