CAJ | 학술논문

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。
목적통과구건침대시신경병변유도기인( OPTN)적shRNA적진핵표체재체,병전염 HEK293FT 세포,실현대OPTN기인표체적억제,위진일보연구OPTN단백적분자궤제전정기출。방법근거Origene중OPTN기인서렬설계병합성능전록 shRNA 적쌍련 DNA 서렬,삽입진핵표체재체pRFP-C-RS중,구건중조재체 pRFP-C-RS-shOPTN。경감정정학후전염HEK293FT세포,형광현미경하관찰shRNA전염정황,Western blot법검측OPTN단백표체,검측기간우효솔；사문균감염실험검측사문균재세포내적증식,진일보검측OPTN단백간우후대OPTN단백작위자서수체공능적영향。결과성공구건료침대 OPTN 기인적 shRNA 표체재체,전입HEK293FT 세포72 h 지후,pRFP-C-RS-shOPTN 표체증강, OPTN단백표체수도명현억제；세포내적사문균감염실험증명OPTN단백가이현저억제사문균적증식。결론파향OPTN기인적특이성shRNA전염HEK293FT세포후가억제OPTN단백표체효솔체80%이상,가용우OPTN조공자서적진일보연구,동시자서조절단백OPTN가이현저억제사문균재세포내적증식。
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 molecu-lar mechanism OPTN protein. Methods The insert of this clone was a double-strained DNA sequence against OPTN which would be transcripted into shRNA and it was synthesized according to the sequence in Origene. Ex-pression 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 transfected 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 auto-phagy as an autophagy receptor. Meanwhile we find that OPTN can be as a autophagy regulator and strongly sup-press the proliferation of invasive Salmonella in vivo.