Pokemon表达慢病毒载体的构建及鉴定

Establishment and Identification of Lentivirus with Pokemon Expression

目的构建Pokemon表达慢病毒载体。方法通过PCR扩增Pokemon cDNA,将Pokemon cDNA连接于GV165载体,经测序确认后,将GV165/Pokemon与pHelper 1.0和pHelper 2.0共转染至293T细胞中,收获病毒,通过Real time PCR测定滴度;将Pokemon表达慢病毒载体侵染293T细胞通过免疫荧光和Western blot检测Pokemon表达慢病毒载体的转染效率和Pokemon的表达能力。结果在感染Pokemon慢病毒载体293T细胞中能检查到Pokemon-GFP融合蛋白的表达。结论成功构建表达Pokemon的慢病毒载体。

Objective To obtain Lentivirus with Pokemon expression. Methods Pokemon cDNA was obtained from eDNA pool by RTPCR, and the Pokemon eDNA was ligated with GV165 vector; the GV165/Pokemon plasmid con firmed by sequencing was cotranfected with pHelper 1.0 and pHelper 2.0 into 293T cells to obtain Pokemon Lentivirus ; ti ter of Lentivirus with Pokemon expression was assessed by Real Time PCR; the infection efficiency of Pokemon Lentivirus in 239T cells was monitored by using immunomicroscope and the expression of Pokemon was detected by Western blot in Pokemon Lentivirus infected 239T cells. Results Pokemon Lentivirus could express Pokemon in 239T cells. Conclu sion Lentivirus with the expression of Pokemon was successfully established.