Fas靶向RNA干扰逆转录病毒载体的构建及生物活性鉴定

Construction of FAS-Targeted RNAi-Retroviral Vector and Its Identification for biological activity

本研究构建Fas靶向RNA干扰逆转录病毒载体,为探讨抑制Fas表达在再生障碍性贫血等疾病治疗中的意义、以及为促进RNA干扰技术的广泛开展奠定基础。用PCR方法获得小鼠U6+27启动子盒及产生siRNA的相应DNA模板,然后连同增强绿色荧光蛋白(EGFP)基因克隆入商业化逆转录病毒载体pLXSN,以获得逆转录病毒载体穿梭质粒pLXSN/EGFPsiFas。包装、滴定重组逆转录病毒载体,并感染P815细胞,检测绿色荧光蛋白表达情况及对细胞Fas表达的抑制情况。结果表明:构建的逆转录病毒载体pLXSN/EGFPsiFas能够有效被包装并感染靶细胞,在靶细胞内可同时表达siRNA、绿色荧光蛋白以及新霉素抗性。结论:成功构建了抑制小鼠Fas表达的RNA干扰逆转录病毒载体。

This study was aimed to construct mouse Fas-targeting si RNA-expressing recombinant retroviral vector in order to explore the therapeutic potential of Fas inhibition by siRNA in the treatment of aplastic anemia and to provide a basia for extensive development of RNA interference techinque, The U6^ 27 promoter cassette and siFas sequence were obtained by PCR method, The U6-siFas fragment was cloned into the multiple restriction site of pLXSN-EGFP and directly downstream of EGFP gene, The resultant retroviral vector pLXSN/EGFP-siFas was packaged using PA317 cell line and tittered using NIH3T3 cell line, P815 cells were infected by the retroviral vector. EGFP expression in P815 was observed under fluorescent microscope and Fas inhibition effect was detected by immunohistochemistry. The results indicated that successfully constructed retrovirus vector pLXSN/EGFP-siFas was could not only deliver siRNA into mammalian cells efficiently and inhibit Fas expression in P815 cells, but also could express EGFP as marker and neomycine resistance gene to allow antibiotic selection. It is concluded that the successful construction of this retroviral vector would greatly facilitate the application of RNA interference and lay the foundation for therapeutic study of Fas inhibition in the treatment of aplastic anemia.