中文摘要
本课题克隆了4株不同亚型AIV即H5N1，H5N2，H7N1，H9N2的全基因并进行序列测定，设计和制备用于检测AIV的基因芯片。利用制备的芯片分别检测高低致病性H5亚型以及低致病性H7、H9亚型AIV在动物体内（肺脏和大脑）和体外（哺乳动物细胞-MDCK和禽类细胞-CEF）上的表达差异，并用real-time RT-PCR方法进行验证。结果表明：我们克隆到的4个毒株的32个基因片段均含有相应病毒基因的完整开放阅读框架；由血凝实验结果和间接免疫荧光实验结果表明这4株病毒在MDCK和CEF细胞上都能够复制；H5N1毒株感染MDCK和CEF细胞后6h，12h，24h各基因表达水平很低，而48h和72h时表达水平较高；H5N2，H7N1，H9N2毒株感染CEF和MDCK细胞后各基因表达水平很低，72h略有表达；体内实验发现H5N1毒株的HA、NA两个外部基因在SPF鸡肺脏和大脑中表达水平比其他3株病毒高，而其他3株病毒间无明显差别，这4株病毒的M等内部基因表达无明显差异。本研究顺利完成了预期研究内容，达到预期目标。
英文摘要
This study cloned and sequenced the whole genes of four strains AIVs,those were H5N1，H5N2，H7N1，H9N2 subtypes seperately. We designed and made genechips to detect AIVs.Using those chips,we detected expression level of different pathogenesis H5 subtypes and low pathogenesis H7 and H9 subtypes AIVs on lung and brain in vivo, mammalian cell and avian cell in vitro.Real-time RT-PCR method was used to identify the results of gene chips. The results showed that thirty-two genes of four strains AIVs we cloned all contain full opening frame;hemagglutinin test and indirect immunofluorescence results showed that four strains viruses all could replicate on mammalian cell-MDCK and avian cell-CEF;The genes of H5N1 strain expressed lowly at 6h,12h,24h after it infected MDCK and CEF while highly expressed at 48h and 72h. H5N2,H7N1,H9N2 strains had low expression at 6h,12h,24h,48h ,but a little highly at 72h. The experiment in viro discovered that HA,NA genes of H5N1 strain expressed more highly than those of other three strains viruses,while other three strains had no significant difference.The inner genes like M gene of the four strains AIVs expressed similiarly.This study completed the all contents anticipated.
结题摘要
本课题克隆了4株不同亚型AIV即H5N1，H5N2，H7N1，H9N2的全基因并进行序列测定，设计和制备用于检测AIV的基因芯片。利用制备的芯片分别检测高低致病性H5亚型以及低致病性H7、H9亚型AIV在动物体内（肺脏和大脑）和体外（哺乳动物细胞-MDCK和禽类细胞-CEF）上的表达差异，并用real-time RT-PCR方法进行验证。结果表明：我们克隆到的4个毒株的32个基因片段均含有相应病毒基因的完整开放阅读框架；由血凝实验结果和间接免疫荧光实验结果表明这4株病毒在MDCK和CEF细胞上都能够复制；H5N1毒株感染MDCK和CEF细胞后6h，12h，24h各基因表达水平很低，而48h和72h时表达水平较高；H5N2，H7N1，H9N2毒株感染CEF和MDCK细胞后各基因表达水平很低，72h略有表达；体内实验发现H5N1毒株的HA、NA两个外部基因在SPF鸡肺脏和大脑中表达水平比其他3株病毒高，而其他3株病毒间无明显差别，这4株病毒的M等内部基因表达无明显差异。本研究顺利完成了预期研究内容，达到预期目标。