人骨髓间质干细胞ING4基因的克隆及其慢病毒表达载体构建

Cloning of human ING4 drived from MSC and construction of PNL-ING4 Lentiviral vector

目的:克隆人骨髓间质干细胞ING4(inhibitor of growth famility,member4)基因,构建其慢病毒表达载体PNL-ING4。方法:提取人骨髓间质干细胞(hMSCs)总RNA,经RT-PCR扩增出ING4cDNA,克隆至PMD19-T载体,选择阳性克隆进行酶切鉴定和测序,构建慢病毒表达载体PNL-ING4,用双酶切、基因测序进行鉴定。结果:RT-PCR产物为750 bp的条带,双酶切和基因测序正确。结论:成功从hMSCs克隆了ING4基因并成功构建其慢病毒表达载体PNL-ING4,为进一步研究ING4基因的作用与抗肿瘤机制奠定了基础。

Objective： To clone the gene of human ING4 derived from human bone marrow-derived mesenchymal stem cells（hMSC） and construct PNL-ING4 lentiviral vector. Methods： The cDNA of ING4 was amplified by RT-PCR using the total RNA extracted from hMSC. The PCR product was inserted into PMD19-T vector. The positive recombinant clone was analyzed by digestion of restriction endonuclease and DNA sequencing. The correct sequence was subcloned into Lentiviral vector. The identification was performed by analysis of restricting enzyme digestion and DNA sequence. Results： RT-PCR product was a 750bp specific fragment. Restriction enzyme digestion and DNA sequencing revealed that ING4 cloning was successful. Conclusion： The gene encoding human ING4 derived from MSC and PNL-ING4 lentiviral vector were obtained, which laid a basis for further researching on its function and tumor suppress mechanisms.