人 SND1基因慢病毒载体构建及稳定表达SND1蛋白的人卵巢癌 SKOV3细胞株筛选.

Objective To construct the lentivirus vector of human SND1 gene and to screen the human ovarian cancer cell line SKOV3 which can stably expresses SND1 protein. Methods The fragment of FLAG-SND1 was cloned from pC-MV-FLAG-SND1 plasmid, and then was inserted into lentivirus vector pLVX-IRES-Hyg. The lentivirus expression vector pLVX-FLAG-SND1 was established and was transiently transfected into 293T cells for 48 h. Western blotting was employed to determine SND1 expression. Then the lentivirus expression vector was co-transfected into 293T cells with packaging plas-mids to obtain the recombinant lentivirus carrying FLAG-SND1.The lentivirus was collected to infect SKOV3 cells. After 48 h of infection, 1 μg/mL hygromycin B was used to screen the infected cells, so as to obtain a cell line with stable ex-pression of SND1 protein. The expression level of SND1 was detected by Western blotting. Results Double restriction en-zyme digestion and DNA sequencing demonstrated the lentivirus expression vector pLVX-FLAG-SND1 was constructed. The recombinant plasmids were transiently transfected into 293T cells, and the expression level was significantly higher than that of the wild-type SKOV3 cells (P<0.01).In the SKOV3 cells which stably expressed SND1 after infection and drug screening, the expression of SND1 was significantly higher than that of the wild-type SKOV3 cells (P<0.01).Conclusion The SND1 gene lentivirus vector pLVX-FLAG-SND1 is constructed successfully and we screen the SKOV3 cell line stably overexpressing SND1 via lentivirus system. [ABSTRACT FROM AUTHOR]