反义HSP70真核表达载体的构建和在人喉癌细胞的表达

目的　进行促凋亡治疗喉癌实验 ,构建和鉴定携带反义HSP70 (heatshockprotein 70 )基因的真核表达载体。方法　将HSP70cDNA反向克隆入 pcDNA 3.1,构建CMV启动子控制的真核表达载体 pcDNA AHSP70 ,用酶切鉴定结果 ;应用该载体转染人喉表皮样癌细胞Hep 2 ,G4 18筛选阳性克隆 ;westernblot和免疫组化检测转染前后瘤细胞的HSP70的表达。检测转染前后瘤细胞的生物学性状。结果　获得 pcDNA AHSP70真核表达载体 ,HSP70反义RNA阻断了Hep 2细胞的HSP70的表达 ,经westernblotting和免疫组化证实 ,实验组瘤细胞不能表达或低表达HSP70 ,而对照和空载体组高表达HSP70。转染反义HSP70的真核表达载体的Hep 2细胞比空载体组和对照组的细胞生长缓慢 ,细胞周期可见实验组出现亚二倍凋亡峰 ,而空载体组没有。结论　本研究成功构建了反义HSP70真核表达载体并在人喉癌细胞得以表达。

Construction and identification of antisense HSP70 eukaryotic expression vector

Objective To construct and identify eukaryotic expression vector carrying human antisense HSP70 cDNA for curing human laryngeal cancer with promoting apoptosis method. Methods HSP70 cDNA was inserted into polylinker sitr of eukaryotic expression vector pcDNA 3.1 to construct pcDNA AHSP70, in which restriction enzyme analysis was used to confirm the reverse orientation of the HSP70 cDNA from the individual transformants. The vector was transfected into human laryngeal cells and the positive clone was screened by G418. The HSP70 expression of Hep 2 cells before and after transfection was detected by western blotting and immunohistochemistry. The biological characteristics of Hep 2 before and after transfection was inspected. Results The pcDNA AHAP70 vector was obtained. HSP70 expression of Hep 2 cells was blocked by antisense HSP70 RNA. It was verified by western blotting and immunohistochemistry that the antisense group showed no or low HSP70 expression, compared with control group. Flow cytometry (FCM) analysis showed subploidy peaks found in the antisense group but not in control group.Conclusion The study makes successful construction and expression of antisense HSP70 eukaryotic expression vector laryngeal cancer cell.