人CD59 RNAi逆转录病毒载体系统的构建与鉴定

CONSTRUCTION AND IDENTIFICATION OF RETRO RNAi SYSTEM OF THE SPECIFIC SILENCING CD59

目的利用siRNA表达载体法构建并筛选携带针对CD59基因pSUPER retro RNAi逆转录病毒载体及稳定产毒的细胞克隆。方法用DNA重组技术,将3条60bp能转录产生靶向CD59小发夹RNA(shRNA)的寡核苷酸序列定向克隆入逆转录病毒载体pSUPER retro,脂质体法转染包装细胞系Phoenix A,建立产生逆转录病毒的细胞克隆。结果重组载体经PCR及限制性内切酶酶切鉴定初步成功后测序序列正确;重组载体转染包装细胞,可表达绿色荧光蛋白,表明包装成功。结论特异性沉默CD59基因的pSUPER retro RNAi逆转录病毒载体以及稳定产毒的细胞系构建成功,为后续进行肿瘤细胞的研究奠定了基础,可望为肿瘤治疗开辟新途径。

Objective To construct recombinant retroviral vectors that target CD59 gene and a stable virus-producing cell line. Methods Three 60 bp encoded targeting CD59 gene shRNA sequences were cloned into pSUPER with DNA recombinant technique. The packaging cell Phoenix A was transfected with this recombinant plasmids using liposome. Results The result of electrophoresis of PCR, enzyme digestion and sequence demonstrated that 60 bp had been inserted into the vector. And virus-producing cell lines were selected. Conclusion Recombinant retroviral vectors and stable virus-producing packaging cell lines are successfully constructed and identified, which opens up a way for studying the role of CD59 in the immune escape of tumor cells and in tumor therapy.