小鼠B7-DC胞外段原核表达载体的构建及表达

目的: 构建小鼠B7-DC胞外段基因的原核表达载体, 并在E.coli BL21中诱导表达.方法: 从小鼠骨髓来源的未成熟树突状细胞(DC)中提取总RNA, 经RT-PCR扩增B7-DC胞外段, 并将其克隆至原核表达载体pET32a( )中, 构建重组表达质粒pET32a( )-B7-DCECD.重组质粒经双酶切鉴定及序列测定后, 转化E.coli BL21, 经IPTG诱导表达目的蛋白, 并用SDS-PAGE和Western blot进行检测.结果: 获得全长为582 bp的小鼠B7-DC胞外段基因, 经测序证实其序列正确.SDS-PAGE和Western blot分析证实重组质粒可表达出Mr约为41 000的B7-DC胞外段蛋白.结论: 成功地构建了小鼠B7-DC胞外段基因原核表达载体, 并在E.coli BL21中进行表达, 为进一步研究B7-DC的功能奠定实验基础.

Construction and expression of prokaryotic vector encoding extracellular region of murine B7-DC

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.