[Construction and expression of prokaryotic vector encoding extracellular region of murine B7-DC].
OBJECTIVE
To construct a prokaryotic expression vector for the extracellular domain of murine B7-DC(B7-DC(ECD)) gene, and to express the gene in E.coli BL21.


METHODS
The total RNA was extracted from murine immature bone marrow-derived dendritic cells and the extracellular fragment of B7-DC cDNA was amplified by RT-PCR. The recombinant plasmid pET32a(+)-B7-DC(ECD) was constructed by cloning the extracellular fragment of B7-DC cDNA into the prokaryotic expression vector pET32a(+). After the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing, pET32a(+)-B7-DC(ECD) was transformed into E.coli BL21 through IPTG induction to express the target protein, and the protein was analyzed by SDS-PAGE and Western blot.


RESULTS
A 582 bp of extracellular fragment B7-DC cDNA was obtained and the sequence was confirmed right 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 fragment of B7-DC is successfully cloned into pET32a (+) and expressed in E.coli BL21, which lays a foundation for the further functional research of B7-DC.
Fan
Yan
Y
School of Life Science, East China Normal University, Shanghai 200062, China. yanfei0016@sina.com
Jiang
Wen-zheng
WZ
Zhang
Ya-li
YL
Wen
Jie-jun
JJ
Hao
Wen-li
WL
Qian
Min
M
D000818:Animals; D018122:B7-1 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D061027:Programmed Cell Death 1 Ligand 2 Protein; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018122:B7-1 Antigen; C556978:Pdcd1lg2 protein, mouse; D061027:Programmed Cell Death 1 Ligand 2 Protein