人B7-H4慢病毒表达载体的构建

目的构建人B7-H4慢病毒表达载体。方法将RT-PCR扩增的B7-H4基因全长编码序列克隆到慢病毒转移载体,通过脂质体转染293细胞,包装成人B7-H4慢病毒颗粒。RT-PCR、流式细胞术和Western blot鉴定B7-H4在重组慢病毒感染293细胞中表达,50%组织培养感染剂量法（TCID50法）检测重组慢病毒滴度。结果成功构建了pMD18-T-hB7-H4质粒和pEZ-Lv105-hB7-H4质粒。基因测序分析证实人B7-H4编码序列成功整合到质粒载体。TCID50法测定Lenti-hB7-9H4慢病毒滴度为1.7×10^9 copies/mL。RT-PCR、流式细胞术和Western blot证实Lenti-hB7-H4慢病毒转染细胞后可有效表达人B7-H4 mRNA和蛋白质。结论成功构建了表达人B7-H4的慢病毒颗粒。

Construction of human B7-H4 lentiviral expression vector

Objective To construct and identify a human B7-H4 lentiviral expression vector. Methods The full-length coding sequence of B7-H4 gene was amplified by RT- PCR, and then cloned into a lentivirus vector. The B7-H4 expression lentiviral particles were packaged after the vector was transfected into 293 cells by liposome. Expression of B7-H4 in 293 cells was identified by RT-PCR, flow cytometry and Western blot, and recombinant lentiviral titer was detected by 50% tissue culture infective dose（TCID50）. Results The pMD18-T-hB7-H4 and pEZ-Lv105-hB7-H4 plasmids were successfully constructed. Gene 9sequencing confirmed the integration of hB7-H4 coding sequence into the plasmid. Lenti-hB7-H4 lentivirus titer was 1.7×10^9 copies/mL. RT-PCR, flow cytometry and Western blot showed expression of human B7-H4 mRNA and protein after Lenti-hB7-H4 lentivirus transfection. Conclusion The human B7-H4 lentiviral expression vector has been successfully constructed.