利用反向遗传技术制备重组人类偏肺病毒  

Generation of Human Metapneumovirus Using Reverse Genetics Technique

作　　者：张金辉[1] 葛金英[2] 任妍[1] 步志高[2] 赵晓东[1] 

机构地区：[1]重庆医科大学附属儿童医院儿研所P2实验室,重庆400014 [2]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001

出　　处：《微生物学通报》2009年第8期1239-1243,共5页Microbiology China

基　　金：国家自然科学基金重点资助项目(No.30730098)

摘　　要：利用反向遗传技术,将包含人偏肺病毒2个血清型A和B代表株NL/1/00和NL/1/99全基因组cDNA的质粒及其4种辅助蛋白N、P、L、M2.1的表达质粒pCITE-N、pCITE-L、pCITE-P、pCITE-M2.1分别共转染BSR-T7细胞以制备重组hMPV。3μg全长cDNA质粒,辅助蛋白质粒pCITE-N、pCITE-L、pCITE-P、pCITE-M2.1分别为0.3μg、0.15μg、0.3μg和0.24μg,通过Lipofectamine2000转染BSR-T7细胞,转染后3天,将BSR-T7细胞于?80°C冻融一个循环以裂解细胞,取上清液感染VeroE6细胞。接种后的VeroE6细胞,6天细胞可观察到明显的细胞病变效应;RT-PCR检测出符合预期目标大小的450bp条带;间接免疫荧光实验检测感染后的VeroE6细胞可在荧光显微镜下可观察到特异性绿色荧光,结果一致表明全基因组cDNA及其辅助质粒产生了有感染性的重组hMPV病毒。用细胞病变法和测试重组病毒的病毒滴度和生长曲线,重组病毒6天时A型代表株NL/1/00在VeroE6细胞滴度为106.4TCID50/mL,B型代表株NL/1/99为105.33TCID50/mL。The full length cDNA clones for hMPV NL/1/00 and NL/1/99 strains from both subtypes, and the four helper vectors pCITE-N, pCITE-L, pCITE-P and pCITE-M2.1 for expressing major viral proteins were co-transfected into BSR-T7 cell to rescue live virons by reverse genetics technique. BSR-T7 cells were transfected using Lipofectamine 2000 with 3 μg of the full-length cDNA plasmid, 0.3 pg of pCITE-N, 0.15 μg of pCITE-L, 0.3 μg of pCITE-P, and 0.24μg of pCITE-M2.1. Three days after transfection, cells were subjected to one -80℃ freeze-thaw cycle to prepare lysates. The lysate was used to inoculate Vero E6 cells. The obvious CPE in Vero E6 cells was observed 6 days after inoculation. The 450 bp RT-PCR products was accordant with the expectant. The specific immunofluorescence was observed for inoculated positive cells.The results demonstrated that infectious hMPV was successfully rescued. The NL/1/00 recombinant virus grew to peak titer of 10^6.4 TCID50/mL in Vero-E6 cells at 6 days after inoculation and 10^5. 33 TCID50/mL for recombinant NL/1/99 virus.

关 键 词：反向遗传技术 人偏肺病毒 病毒拯救 

分 类 号：R346[医药卫生—基础医学]