RNAi沉默Fas受体表达的腺病毒载体构建

Construction of Recombinant Adenovirus Vector expressing Two Fas-Targeted shRNA

目的:利用pEGFP6-1和pGenesil-1质粒构建针对小鼠Fas的2个短发卡状RNA(short hairpinRNA,shRNA)串联表达的载体pEGFP6-1-siFas1+siFas2,进一步通过BDAdeno-X系统构建腺病毒表达Fas-shRNA载体。方法:设计表达2个短发卡状RNA结构的互补DNA序列分别克隆至质粒载体pEGFP6-1和pGenesil-1中,酶切pEGFP6-1-siFas1大片段和酶切pGenesil-1-siFas2小片段,经T4DNALigase连接得到pEGFP6-1-siFas1+siFas2重组质粒。双酶切重组质粒和pShuttle2穿梭质粒,经T4DNA连接酶连接得到pShuttle2-siFas1+siFas2质粒。双酶切重组穿梭质粒,连接酶切产物和腺病毒转化感受态E.coilDH5а,PCR筛选鉴定阳性克隆,进一步线性化重组腺病毒pAdeno-siFas1+siFas2质粒,经脂质体转染HEK293A细胞;收细胞进行裂解收病毒。结果:构建的重组质粒载体经酶切鉴定和测序分析,shRNA编码序列与设计的片段完全一致;重组腺病毒载体经酶切鉴定和PCR分析正确;经HEK293A细胞包装成功,能够表达绿色荧光蛋白。结论:成功构建Fas-shRNA腺病毒串联表达载体。

Objective： To construct the recombinant adenovirus vector expressing two Fas - targeted short hairpin RNA （shRNA） by pEGFP6 - 1 - siFasl ＋ siFas2. Methods：Two pairs of DNA sequences were designed and synthesized to form complementary chains by annealing respectively. Then the obtained products were inserted ifito plasmid vector pEGFP6- 1/pGenesil- 1 with U6 promoter. The two plasmids were first digested to completion with Sac1, followed by Sail digestion. A long segment from pEGFP6 - 1 - siFas 1 and a short segment from pGenesil- 1 - siFas2 were reclaimed, and connected with T4 DNA Ligase. The pEGFP6 - 1 - siFas 1 ＋ siFas2 and pShuttle2 were digested ment from pEGFP6 - 1 - siFas 1 ＋ si restriction endonuclease. A long segment from pShuttle2 and a short seg- 2 were reclaimed, and connected. Then the recombinant plasmid were digested to completion with PI - Sce Ⅰ and Ⅰ - Ceu I . A 2 706 bp segment from pShuttle2 - siFas 1 ＋ siFas2 and BD Adeno - X Viral DNA were connected. The recombinant plasmid series pAdeno - siFasl ＋ siFas2 were digested by Pac I completely. Linear pAdeno - siFasl ＋ siFas2 were transformed into HEK293A cell. The adenovirus vector gets packed, and then the adenovirus was reclaimed basing on present of the Cytopathic Effect. iResults： The two Fas - targeted shRNAs expressing frames were successfully inserted into the plasmid vector pEGFP6 - 1. The adenovirus vector with two Fas - targeted shRNAs expressing frames was successfully construted. The adenovirus gets packed correctly in HEK293A cell. Conclusion：The recombinant adenovirus vector with two Fas - targeted shRNAs was successfully constructed and packed.