Desmuslin基因逆转录病毒表达载体的构建及包装细胞株的建立

Construction of desmuslin gene retroviral vector and the establishment of its package cell lines

目的构建Desmuslin（DMN）基因的逆转录病毒表达载体，并建立其包装细胞株。方法聚合酶链式反应（PCR）扩增DMN基因的全长编码框（约3700bp），PCR产物（PCR—DMN）亚克隆至逆转录病毒载体pRetroQ—AcGFP1-C1，构建DMN基因的逆转录病毒表达载体pRetroQ—AcGFP1-C1-DMN，酶切及测序鉴定后，把重组质粒通过转染导人包装细胞株PT67，嘌呤霉素筛选后获得抗性克隆，293细胞进行病毒颗粒滴度测定。结果pRetroQ—AcGFP1-C1-DMN克隆的酶切鉴定和测序鉴定结果表明DMN基因的全长编码框被准确克隆至载体pRetroQ—AcGFP1-C1，其序列与理论序列完全-致；获取了稳定产生DMN逆转录病毒的PT67细胞克隆株，其产生的病毒原液平均病毒滴度为（2．80±1．46）×10^6cfu／ml。结论成功构建了Desmuslin基因逆转录病毒表达载体并建立了其包装细胞株。

Objective To construct desmuslin （DMN） gene recombinant retroviral vector and es- tablish its package cell lines. Methods DMN gene （-3700 bp） was amplified by using polymerase chain reaction （PCR） and cloned into retroviral vector （pRetroQ-AcGFP1-C1）. Recombinant retroviral vector pRetroQ-AcGFP1-C1-DMN was constructed and identified by sequencing, and transfected into PT67 pack- age cells by LipofectamineTM LTX and PLUSTM. After 48 h, cells stably expressing pRetroQ-AcGFP1-C1- DMN retroviral vector were screened by puromyein, and the viral titer was detected by infecting 293 cells. Results The recombinant retroviral vector pRetroQ-AeGFP1-C1-DMN was successfully constructed, and the cloning site and reading frame of the objective gene were confirmed by enzyme digestion and sequen- cing. PT67 package cell lines were successfully established and average viral titer was （ 2. 80 ± 1.46 ） × 106 cfu/mL. Conclusion DMN gene retroviral vector has been successfully constructed and its package cell line PT67-DMN has been established.