中文摘要：

目的建立慢病毒介导且稳定表达病毒白细胞介素6（viral interleukin-6,vIL-6）蛋白的血管内皮细胞株。方法以质粒pvIL-6-Flag为模板,PCR扩增vIL-6基因,克隆至慢病毒载体p3DLV,经酶切和测序鉴定后,将重组质粒与慢病毒辅助包装元件质粒共转染293T细胞,构建含vIL-6基因的重组慢病毒。重组慢病毒感染血管内皮细胞EA.hy926,经潮霉素筛选,获得稳定表达细胞株,western blot鉴定vIL-6蛋白的表达。结果酶切鉴定和基因测序证实长度为615 bp的vIL-6基因成功克隆至慢病毒表达载体;重组慢病毒经包装、纯化后测得滴度为1.0×107 TU/mL。重组慢病毒感染EA.hy926细胞,经潮霉素筛选,形成生长形态良好的单克隆细胞株EA.hy926-vIL-6。western blot鉴定该细胞株可稳定表达vIL-6蛋白。结论建立了稳定表达vIL-6的EA.hy926细胞株,为进一步研究vIL-6的生物学功能及致病机制提供了细胞模型。

英文摘要：

Objective To establish a vascular endothelial cell line stably expressing virus IL-6 （vlL-6） gene transduced by lentivirus. Methods The vlL-6 gene was amplified from plasmid pvIL-6-Flag by PCR. The purified vIL-6 gene fragment was inserted into lentivirus vector （p3DLV） and the recombinant plasmid was identified by restriction enzyme digestion and DNA sequencing. 293T cells were cotransfected with the recombinant plasmid and the helper plasmids, thus the recombinant lentivirus carrying vlL-6 was successfully packaged. The recombinant lentivirus was transfected the vascular endothelial cells EA. hy926, and the resistant cell clones were se- lected with hygromycin. The expression of vIL-6 was examined using western blot. Results Enzyme digestion and DNA sequencing showed that the full-length vIL-6（615 bp） gene was successfully subcloned into the lentiviral vector. After transfection, the recombinant lentivirus was packaged into 293T cells. The titer of recombinant lentivirus was 1.0 × 10^7 TU/mL. The monoclonal cell line EA. hy926-vIL-6 was produced after transfection with the recombinant lentivirus and selected with hygtomycin. The stable expression of vIL- 6 protein was verified by western blot. Conclusion An EA. hy926 cell line stably transduced with lentivirus carrying vlL-6 gene was successfully generated. It can be used as a cell model to study the biological function and pathogenesis of vIL-6.