Wang Xue-feng, He Yuan-li
Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2008 Oct;28(10):1856-9.
To construct a lentiviral vector carrying human bcl-2 gene and investigate its expression in human ovarian granulosa cells (GCs).
Human bcl-2 gene was amplified from the plasmid pCMV-SPORT6 using PCR and subcloned into the lentiviral vector pGC-FU to construct the lentiviral expression vector pGC-FU- bcl-2. The bcl-2 gene insert was confirmed by restriction enzyme digestion and sequencing. The recombinant lentiviruses generated by 293T cells co-transfected with pGC-FU-bcl-2 and the packaging plasmids pHelper1.0 and pHelper2.0. The resulting recombinant lentivirus GC-FU-bcl-2 carrying bcl-2 and EGFP genes were then used to infect human ovarian granulosa cells. EGFP and bcl-2 protein expressions in 293T and human ovarian GCs were detected by fluorescent microscope and Western blotting.
The plasmid pGC-FU-bcl-2 carrying the correct bcl-2 gene sequence could be expressed in human ovarian GC cells, and the recombinant lentivirus GC-FU-bcl-2 was generated by the packaging 293T cells. Stable expression of EGFP and bcl-2 proteins were detected by fluorescent microscope and Western blotting in 293T and human ovarian GCs after the infection. The recombinant lentivirus efficiently delivered bcl-2 gene into human ovarian GCs, in which bcl-2 expression was expressed efficiently and stably.
The recombinant lentivirus GC-FU-bcl-2 has been successfully constructed, which is capable of delivering the target gene bcl-2 into human ovarian GCs for its stable expression.
构建携带人bcl-2基因的慢病毒载体，并研究其在人卵巢颗粒细胞（GCs）中的表达。
采用PCR从质粒pCMV-SPORT6中扩增人bcl-2基因，并亚克隆至慢病毒载体pGC-FU，构建慢病毒表达载体pGC-FU-bcl-2。通过限制性内切酶酶切和测序确认bcl-2基因插入情况。用pGC-FU-bcl-2与包装质粒pHelper1.0和pHelper2.0共转染293T细胞产生重组慢病毒。然后用携带bcl-2和EGFP基因的重组慢病毒GC-FU-bcl-2感染人卵巢颗粒细胞。通过荧光显微镜和蛋白质免疫印迹法检测293T细胞和人卵巢颗粒细胞中EGFP和bcl-2蛋白的表达。
携带正确bcl-2基因序列的质粒pGC-FU-bcl-2可在人卵巢颗粒细胞中表达，包装293T细胞产生了重组慢病毒GC-FU-bcl-2。感染后，通过荧光显微镜和蛋白质免疫印迹法在293T细胞和人卵巢颗粒细胞中检测到EGFP和bcl-2蛋白的稳定表达。重组慢病毒有效地将bcl-2基因导入人卵巢颗粒细胞，其中bcl-2表达高效且稳定。
成功构建了重组慢病毒GC-FU-bcl-2，其能够将靶基因bcl-2导入人卵巢颗粒细胞并实现稳定表达。
