人α-防御素基因的克隆及其在293T细胞中表达的临床意义

Cloning of humanα-defensin gene and its expression in 293T cell

目的克隆人α-防御素(α-HNP)基因,构建重组真核表达质粒pCG-HNP并检测其在293T细胞中的表达。方法提取人PBMC细胞的RNA,以其为模板用RT-PCR技术克隆人α-HNP的cDNA,并将其插入质粒pCG中构建真核表达载体。测序证实后,将重组质粒用脂质体法转染293T细胞,用蛋白质免疫印迹法(Western)检测目的蛋白分子的表达。结果测序证实克隆的人α-HNP阅读框正确完整,PCR鉴定和酶切鉴定证实插入方向正确,免疫印迹结果显示重组质粒能够在293T细胞中表达HNP-GFP-Flag融合蛋白。结论α-HNP基因成功克隆;其在293T细胞中的表达可为进一步研究其抗HIV活性奠定基础。

[ Objective] To clone the human α-HNP and to construct the recombinant eukaryotic expression plasmid pCG-HNP for expression in 293T cells. Method： Using human PBMC RNA as template, we cloned the ORF of human α- HNP by RT-PCR. The ORF was inserted into plasmid pCG for construction of the eukaryotie expression vector, which subsequently was transfeeted into 293T cells after sequencing by lipid transfeetion method. The expression of HNP-GFP-Flag in 293T cells was detected by western blotting. [ Results] Sequencing confirmed that the ORF of the human α-HNP was intact and right. PCR identification and restrictive enzyme digestion proved that the ORF was inserted into pCG in correct direction. Determination with Flag antibody by western blotting, an antieipative band by the cell lysate was displayed, but not in control. [ Conclusion ] The human α-HNP has been cloned successfully and it was found to be expressed in 293T cells.