HSP90β干扰和过表达慢病毒载体的构建及其在骨肉瘤细胞Saos-2中的表达

目的:获得热休克蛋白90β(HSP90β)基因干扰和过表达慢病毒表达系统，并检测其在人骨肉瘤细胞株Saos-2中的表达水平。方法:设计合成shRNA，以慢病毒表达质粒构建HSP90β干扰和过表达载体，酶切电泳、测序技术鉴定载体构建是否成功。重组病毒转染H1299细胞，以嘌呤霉素筛选稳定转染的Saos-2细胞，通过荧光显微镜观察计数获得转染效率。将感染好的细胞分为干扰NC组（NEG-shRNA）、干扰组（shRNA-HSP90β）、过表达NC对照组（NEG-pEZ）及过表达组（pEZ-HSP90β）。通过qRT-PCR 与Western blotting 分别从mRNA 和蛋白表达水平验证目的基因的干扰和过表达水平。结果:插入慢病毒表达载体的基因片段与目的基因的碱基序列完全一致。病毒包装成功后，嘌呤霉素最小致死浓度1 μg/ml，感染复数200，感染人Saos-2的感染效率达80%，其中shRNA-HSP90β组干扰效率为86.35%，pEZ-HSP90β组的mRNA相对表达量增加2.8倍。进一步研究发现，shRNA-HSP90β组较NEG-shRNA组中HSP90β蛋白表达明显降低，pEZ-HSP90β组较NEG-pEZ组HSP90β蛋白表达增加。结论:HSP90β基因干扰和过表达慢病毒载体构建成功，并能够在Saos-2中稳定表达。

Construction of HSP90β interference and overexpression lentiviral vector and its expression in osteosarcoma cell line Saos-2

Objective:To obtain interference and overexpression of HSP90β through lentivirus，and its expression in osteosarcoma cell line Saos-2.Methods:shRNA was designed and synthesized，and HSP90β interference and overexpression vectors were constructed based on lentivirus expression plasmids.PCR and sequencing techniques were utilized to confirm successful construction of vector.The plasmids system was performed to package the virus in H1299 cells.Virus titer was measured by fluorescence microscope.Saos-2 transfected with virus were identified by Puromycin.Transfection efficiency was measured by fluorescence microscope.Further，the infected cell groups were interfered with NC group (NEG-shRNA)，interference group (shRNA-HSP90β)，NC control group (NEG-pEZ) and overexpression group (pEZ-HSP90β).The interference and overexpression levels of target genes were verified by qRT-PCR and Western blotting.Results:The HSP90β gene fragment inserted into the lentivirus expression vector was identical to the target gene.After successful viral packaging，the minimum lethal concentration (MLC) of puromycin was 1 μg/ml and multiplicity of infection (MOI) was 200.The infection efficiency of Saos-2 was 80%.The interference efficiency of shRNA-HSP90β group was 86.35%，and the relative expression of pEZ-HSP90β group increased by 2.8 times.Further studies showed that the expression of HSP90β in shRNA-HSP90β group was significantly lower than that in NEG-shRNA group，and the expression of HSP90β in pEZ-HSP90β group was higher than that in NEG-pEZ group.Conclusion:Interfering and overexpression of HSP90β through lentivirus vector is successfully constructed and could be stably expressed in osteosarcoma cell line Saos-2.