大鼠PEDF44mer慢病毒载体的构建及鉴定

目的 构建大鼠色素上皮衍生因子（PEDF）44mer基因的慢病毒表达载体并检测其表达.方法 化学合成PEDF 44mer-6his,亚克隆入GV206载体,测序鉴定.挑取阳性克隆转染293T细胞,实时荧光定量PCR法测定病毒滴度.将已构建的病毒转染293T细胞,RT-PCR和Western blot法检测44mer-6his表达.结果 阳性克隆测序与设计的44mer基因序列完全一致,44mer慢病毒构建成功.实时荧光定量 PCR法检测病毒滴度为2.00E＋9 TU/ml.44mer慢病毒转染293T细胞后,RT-PCR检测到44mer表达.Western blot显示44mer-6his融合蛋白表达阳性.结论 成功设计构建了PEDF 44mer慢病毒,为进一步研究44mer在缺血性心肌病中的作用和机制奠定基础.

Construction and identification of a lentiviral vector carrying rat PEDF 44mer gene

Objective To construct a lentiviral vector carrying rat pigment epithelium - derived factor （PEDF） 44mer gene and to detect its expression. Methods PEDF 44mer -6his gene obtained by chemical synthesis was sub - cloned into GV206 vectors and undergone sequencing. Positive clones were selected and transfected into 293T cells and virus titers were examined by real - time PCR. Then, the viruses established were transfected into 293T cells. The ex- pression of 44mer - 6his was examined by RT - PCR and Western blot. Results The sequence of the positive clones was completely consistent with that of 44mer gene, suggesting the success of 44mer lentivirus establishment. A virus titer of 2.00E ＋ 9 TU/ml was determined by real -time PCR. The presence of 44mer lentivirus cop~ld be detected by RT- PCR after transfection with 44mer gene lentivirus into 239T cells. The expression of 44mer - 6his fusion protein was detected using Western blott. Conclusion The successful construction of the lentiviral vector carrying rat PEDF 44mer gene laid foundation for further research about the role of 44mer gene in ischemic cardiomyopathy.