【作者】 刘志军；

【导师】 王泽霖；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2004， 硕士

【摘要】 本实验应用RT-PCR技术将河南地方分离株A/Chichen/HN43/98(H9N2)的HA、NA基因片段反转录成cDNA，然后对HA、NA基因的上、下游引物进行磷酸化，以cDNA为模板扩增HA、NA基因，再用T4 DNA 聚合酶处理HA、NA扩增片段。处理用于克隆的质粒载体：用限制性内切酶SapⅠ切开质粒载体pPolⅠSapⅠT，Klenow大片段酶补平，小牛碱性磷酸酶移去5’末端磷酸基团。在T4 DNA连接酶作用下，将处理过的线性载体pPolⅠSapⅠT分别与上述HA、NA基因的cDNA进行体外连接，筛选出阳性重组子pPolⅠHA T、pPolⅠNA T。将pPolⅠHAT、pPolⅠNAT质粒与转染系统的6个转录质粒pPolⅠPAT、pPolⅠPB1T、pPolⅠPB2T、pPolⅠNPT、pPolMⅠT、pPolⅠNST以及4个蛋白质表达重组质粒pcDNA3.0-PA、pcDNA3.0-PB1、pcDNA3.0-PB2、pcDNA3.0-NP共转染293T细胞，然后用转染后的细胞培养液接种9-11日龄SPF鸡胚，收获其尿囊液测的得HA效价为4log2，再经SPF鸡胚连续传3代，HA效价上升至8log2。经血清学及分子生物学方法鉴定，证实了H9N2亚型禽流感病毒的重组成功。本试验以河南地方分离株A/Chichen/HN43/98（H9N2）作为HA、NA基因片段的捐赠株，应用反向遗传技术成功地重组了一株HNP/03（H9N2）亚型禽流感病毒株，不仅为进一步研究H9N2亚型禽流感地方株的毒力变异机制及相关的生物学功能奠定了基础，也为改造和培育优良的禽流感疫苗株提供了可借鉴的模式。更多还原

【Abstract】 In this study , the RNA segments of HA and NA gene were extracted from a donor strain ,A/Chicken/HN43/98(H9N2), which was isolated from Henan province and indentified in 1998 ;then were transcripted reversely into cDNA by RT-PCR technology ,and then the two pairs of primers of HA and NA gene were phosphated respectively . Consequently, HA and NA gene were amplified by the cDNA used as template. Then the segments of HA and NA gene amplified were digested respectively by T4 DNA polymerase . The plasmid vector pPolⅠSap ⅠT were digested by the restriction enzyme Sap Ⅰ and DNA polymerases and so on. At first , The plasmid pPolⅠSap Ⅰwas digested by the restriction enzyme Sap Ⅰ,then was complemented by Klenow DNA polymerase , and then the phosphate group of the 5’ termination was removed by Calf Intestine Alkaline Phospatase (CIAP).Linear plasmid vector pPolⅠSap ⅠT treated linked respectively up the cDNA of HA and NA gene descriped above with the catalysis of T4 DNA ligase in vitro ,then the positive plasmids pPolⅠHAT and pPolⅠNAT were selected respectively.The positive plasmids pPolⅠHAT and pPolⅠNAT reassorted successfully in the study and other vRNAs transcription plasmids of the transfection system as well as the protein plasmids (pcDNA3.0-PB1、<WP=67>pcDNA3.0-PA、pcDNA3.0-NP、pcDNA3.0-PB2) from the transfection system coincubated for an hour with the reagent for transfection ,then cotransfected into near-cofluent 293T cell.Nine-to-11-day-old specific pathogen-free (SPF) embryonated chicken embryos were inoculated with tissue culture fluids of posttransfection , then the allantoic fluids of embryonated-eggs were harvested , HA test was carried out ,and the result was 4log2. Then the reassortant AIV were passaged three times in SPF embryonated chicken embryos. HA test was performed and the result was up to 8log2. The success of reassortment of H9N2 subtype Influenza A Virus was confirmed by the identification of serology and molecular biology.In this experiment , the local isolate A/Chicken /HN43/98 (H9N2) AIV from Henan province was used to be a donor of HA and NA gene , then a HNP/03(H9N2) Influenza A Virus strain was reassorted successfully. This do not only make a basis for further research of the mechanism for virulence variation of H9N2 subtype Influenza A Virus and biological function, but also present a kind of model for the development of good vaccine candidate of Influenza A Virus.更多还原