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目的构建基于RNP复合体的流感病毒反向遗传学操作系统,并利用该系统表达绿色荧光蛋白。方法将人源pol Ⅰ启动子分别插入真核表达载体pcDNA3和原核质粒pUC18,获得pol Ⅰ/pol Ⅱ双启动子表达载体pCHBW和pol Ⅰ单启动子表达载体pPol IR;来源于禽流感病毒H5N1湖北分离株A/Chicken/Hubei/489/2004的基因被克隆到pCHBW上,获得pCHBW-PB2、pCHBW-PB1、pCHBW-PA、pCHBW-NP;将荧光蛋白基因置换NA基因的编码区进而克隆到pPol IR上获得pPol IR-NA(EGFP);这些质粒共转染293T细胞并观察荧光。结果所构建的载体均经酶切和测序验证正确。pCHBW-PB2、pCHBW-PB1、pCHBW-PA、pCHBW-NP和pPol IR-NA(EGFP)共转染293T细胞后能观察到绿色荧光,Western-blot实验证实被转染的293T细胞表达绿色荧光蛋白(EGFP)。结论基于RNP复合体的反向遗传操作系统构建成功,并表达绿色荧光蛋白。
Objective To construct the reverse genetics operating system of influenza virus based on RNP complex and to use this system to express green fluorescent protein. Methods Human pol Ⅰ promoter was inserted into eukaryotic expression vector pcDNA3 and prokaryotic plasmid pUC18 respectively to obtain pol Ⅰ / pol Ⅱ double promoter expression vector pCHBW and pol Ⅰ single promoter expression vector pPol IR; derived from avian influenza virus H5N1 Hubei The gene of isolate A / Chicken / Hubei / 489/2004 was cloned into pCHBW to obtain pCHBW-PB2, pCHBW-PB1, pCHBW-PA and pCHBW-NP. The fluorescent protein gene was substituted for the NA gene coding region and cloned into pPol PPol IR-NA (EGFP) was obtained on IR; these plasmids were co-transfected into 293T cells and fluorescence was observed. Results The constructed vectors were verified by enzyme digestion and sequencing. Green fluorescence was observed after cotransfection of 293T cells with pCHBW-PB2, pCHBW-PB1, pCHBW-PA, pCHBW-NP and pPol IR-NA (EGFP). Western blot analysis confirmed that transfected 293T cells expressed green fluorescent protein (EGFP). Conclusion The reverse genetic operation system based on RNP complex was successfully constructed and expressed green fluorescent protein.