OPTN基因shRNA真核表达载体的构建及干扰效应的检测

Construction of shRNA eukaryotic expression vector targeting OPTN gene and detection of interference effect

目的通过构建针对视神经病变诱导基因(OPTN)的shRNA的真核表达载体,并转染HEK293FT细胞,实现对OPTN基因表达的抑制,为进一步研究OPTN蛋白的分子机制奠定基础。方法根据Origene中OPTN基因序列设计并合成能转录shRNA的双链DNA序列,插入真核表达载体pRFP-C-RS中,构建重组载体pRFP-C-RS-shOPTN。经鉴定正确后转染HEK293FT细胞,荧光显微镜下观察shRNA转染情况,Western blot法检测OPTN蛋白表达,检测其干扰效率;沙门菌感染实验检测沙门菌在细胞内的增殖,进一步检测OPTN蛋白干扰后对OPTN蛋白作为自噬受体功能的影响。结果成功构建了针对OPTN基因的shRNA表达载体,转入HEK293FT细胞72 h之后,pRFP-C-RS-shOPTN表达增强,OPTN蛋白表达受到明显抑制;细胞内的沙门菌感染实验证明OPTN蛋白可以显著抑制沙门菌的增殖。结论靶向OPTN基因的特异性shRNA转染HEK293FT细胞后可抑制OPTN蛋白表达效率达80%以上,可用于OPTN调控自噬的进一步研究,同时自噬调节蛋白OPTN可以显著抑制沙门菌在细胞内的增殖。

Objective To construct shRNA against optineurin （OPTN） clone and transfect the construction into HEK293FT cells for inhibiting the expression of OPTN, which is laid the foundation for further research of molecular mechanism OPTN protein. Methods The insert of this clone was a double-strained DNA sequence against OVFN which would be transcripted into shRNA and it was synthesized according to the sequence in Origene. Expression vector pRFP-C-RS was employed to fuse the insert to get the construction and finally confirmed by sequen- cing. HEK293FT cells were transfected with the very clone pRFP-C-RS-shOPTN and RFP signals could be detected using fluorescence microscope. Western blot was further employed to check the protein level of OPTN. HEK293FT cells were transfeeted with the very clone pRFP-C-RS-shOPTN was then obtained to investigate the functional study of OPTN which was measured by Salmonella infection experiment. Results shRNA eukaryotic expression vector targeting OPTN gene was successfully constructed. HEK293FT cells were transfected with pRFP-C-RS-shOPTN and 72 hours later we observed strong RFP signals showing that shOPTN expressed. Western blot was further employed to check the protein level of OPTN which showed that OPTN expression was indeed interfered. Salmonella infection assays was then performed and showed that OPTN could inhibit the proliferation of Salmonella that has invaded HEK293FT cells. Conclusion HEK293FT cell line which expresses shRNA against OPTN show a sharp inhibition of OPTN protein expression by 80%. So this cell line can be further used to investigate how OPTN regulates autophagy as an autophagy receptor. Meanwhile we find that OVFN can be as a autophagy regulator and strongly sup- press the proliferation of invasive Salmonella in vivo.