摘要
目的构建人组织型基质金属蛋白酶抑制剂2(TIMP-2)编码序列基因逆转录病毒表达载体,转染人单核细胞白血病细胞株,为探讨TIMP-2的生物学功能奠定基础。方法根据基因库中已公布的序列设计引物,用PCR方法扩增出人TIMP-2编码序列基因片段,用限制性内切酶及T4连接酶将目的片段插入MSCV逆转录病毒载体中。采用酶切和PCR鉴定及测序后经脂质体介导转染至PT67包装细胞中,以病毒上清感染单核细胞白血病细胞株SHI-1,G418筛选,极限稀释法挑选单克隆细胞株,定量PCR和Western Blotting检测TIMP-2的表达。结果TIMP-2基因克隆进逆转录病毒载体MSCV中,经酶切、PCR和DNA测序证实重组逆转录病毒载体构建正确,病毒上清感染SHI-1细胞,经G418筛选并挑选出单克隆细胞,经实时定量PCR和Western Blotting检测发现SHI-1细胞中TIMP-2 mRNA及蛋白表达明显上调。结论成功构建了高表达TIMP-2的SHI-1细胞,可对其生物学行为作进一步研究。
Objective To establish a leukemia cell line SHI-1 expressing highly TIMP-2. Methods The human TIMP-2 eDNA was amplied from the ECV 304 cell line by high fidelity PCR, retroviral vector MSCV carrying TIMP-2 eDNA was constructed and the retroviral producer cell line PT67 was developed by liposome-mediated transfection. SHId leukemia cells were transfected by MSCV-TIMP- 2 virus and screened by G418. Cell subelones were isolated by limited dilution. The expression of TIMP-2 of SHI-1/MSCV-TIMP-2 cells was detected by real-time PCR and TIMP-2 protein was analyzed by western-blotting. Results The retroviral vector MSCV-TIMP-2 was successfully constructed and transfected into SHI-1 leukemia cells, and the TIMP-2 gene expression of SHI-1/MSCV-TIMP-2 was up-regulated. Conclusion SHI-1/MSCV-TIMP-2 leukemia cells can be used for the further research on the function of TIMP-2 gene.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2006年第2期185-187,199,共4页
Suzhou University Journal of Medical Science
基金
江苏省卫生厅基金资助项目(H200327)
