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目的:构建携带p73α基因的慢病毒系统表达载体,成功感染人卵巢癌细胞PEO1后检测其蛋白质的表达。方法:将采用PCR技术获得全长p73α基因亚克隆到pCDH-CMV-MCS-EF1-copGFP中,构建慢病毒表达质粒PCDH-p73α,验证成功后与包装质粒pPACKH1-GAG、pPACKH1-REV、Pvsv-G共转293T细胞,获得重组慢病毒Lenti p73α后感染靶细胞人卵巢癌细胞PEO1(p73α基因表达缺失)后,RT-PCR和Western Blotting验证PEO1细胞中Lenti p73α的表达。结果:经酶切鉴定及基因测序证实PCDH-p73α中携带正确的p73α基因序列,与GenBank中序列一致;转染包装细胞293T后产生慢病毒颗粒能有效感染靶细胞PEO1,转导效率约为60%,并且存在p73α基因mRNA和蛋白水平的高表达。结论:本实验成功构建了PCDH-p73α慢病毒表达载体,包装成功后能有效感染卵巢癌PEO1细胞。
OBJECTIVE: To construct a lentiviral vector carrying p73α gene and successfully detect its expression in human ovarian cancer cell line PEO1. Methods: The full-length p73α gene was subcloned into pCDH-CMV-MCS-EF1-copGFP by PCR. The lentiviral expression plasmid PCDH-p73α was constructed and verified by PCR with the plasmid pPACKH1-GAG, pPACKH1-REV, Pvsv- G were co-transfected into 293T cells. The recombinant lentivirus Lenti p73α was transfected into human ovarian cancer cell line PEO1 (whose expression of p73α gene was absent). The expression of Lenti p73α in PEO1 cells was detected by RT-PCR and Western Blotting. Results: The correct p73α gene sequence in PCDH-p73α was confirmed by restriction enzyme digestion and sequencing, which was consistent with the sequence in GenBank. The lentiviral particles 293T after transfection could effectively infect target cell PEO1, and the transduction efficiency was about 60 %, And there is a high expression of p73α gene mRNA and protein levels. Conclusion: The PCDH-p73α lentiviral vector was successfully constructed and successfully infected PEO1 cells.