真核表达载体PcDNA3.1-BDNF的构建及其在骨髓基质细胞中的表达

目的研究基因真核表达载体PcDNA3.1-BDNF的构建及其在骨髓基质细胞的转染和表达情况。方法以挪威大鼠BDNFcDNA为模板,PCR扩增BDNF全长编码基因,亚克隆至PcDNA3.1表达载体。将构建的重组质粒测序并通过脂质体转染到骨髓基质细胞中,RT-PCR鉴定BDNF基因的表达,Western blot检测BDNF蛋白的表达。结果 BDNF基因成功克隆到表达载体PcDNA3.1中,酶切鉴定片段为800bp,Western blot检测到BDNF在骨髓基质细胞呈阳性表达。结论真核表达载体构建成功,BDNF转染BMSCs后可稳定、高效表达。

Construction of PcDNA3.1-BDNF and its expression in bone marrow stromal cells

Objective To construct the recombinant expression plasmid of brain derived neurotrophic factor(BDNF) gene and identify its expression in bone marrow stromal cells(BMSCs).Methods The BDNF coding sequence was amplified by polymerase chain reaction(PCR) method and subcloned into PcDNA3.1 vector.After the target region was sequenced,the plasmid was transfected into BMSCs.The expression of the recombinant plasmid in BMSCs was proved by Western blot.Results PcDNA3.1-BDNF expression vector was constructed successfully,the length of the fragment was 800bp and identified by restriction enzymes digestion.The positive expression of PcDNA3.1-BDNF fusion protein was detected by Western blot in BMSCs.Conclusion The recombinant plasmid of PcDNA3.1-BDNF was constructed successfully and its fusion protein expressed in BMSCs.