Aim: 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.
Conclusion: 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.