【作者】 贾珊珊；

【导师】 王君伟；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 禽流感(Avian Influenza，AI)是由正黏病毒科A型流感病毒引起的传染性疾病综合症。低致病力禽流感常引起呼吸道症状，产蛋量下降。高致病性禽流感，一旦暴发，将会给中国乃至世界很多国家养禽业造成相当严重的经济损失。同H5亚型禽流感相比，H7亚型禽流感的暴发同样会给养禽业造成巨大的损失。H7亚型禽流感病毒不仅能侵袭鸡鸭等家禽，还能使笼养宠物鸟致病(如H7N1型)，甚至能感染人类。AIV基因组为8节段负链RNA，共编码10种与病毒结构和功能相关的蛋白质，其中血凝素(Hemagglutinin，HA)是AIV囊膜纤突的主要成分之一，血凝素(HA)是流感病毒的主要表面糖蛋白，HA具有产生中和抗体的能力，可产生免疫保护作用，因此血凝素在禽流感的诊断和防制中起重要作用。本研究以试验室保存的pMD18-T-H7-HA阳性质粒为模板，去除HA编码信号肽的核苷酸部分，应用生物学软件Primer5.0和Oligo4.0，分别设计HA和HA1两对引物，扩增出HA和HA1基因。并分别将其克隆至昆虫杆状病毒转移载体pBlueBacHis2A的SacⅠ和KpnⅠ之间的多克隆位点上，命名为pBlueBacHis-H7-HA和pBlueBacHis-H7-HA1。纯化重组质粒并与杆状病毒共转染Sf9昆虫细胞，5d后获得重组病毒，命名为P1。将经过3轮蚀斑筛选纯化的重组病毒接种Sf9细胞大量繁殖后收获，PCR鉴定结果为完全纯化的病毒命名为P2。接种P2于Sf9单层细胞上，4d后收获病变细胞，经SDS-PAGE电泳鉴定，表达产物HA、HA1融合蛋白分子量约为66ku和42ku，与理论值相符。表达产物的Westernblotting和Dot-ELISA结果表明其可与鸡抗H7亚型血清发生特异性抗原反应，而与H5和H9亚型鸡抗血清间无交叉反应。血凝试验和间接免疫荧光试验证明了重组HA、HA1蛋白具有良好的生物学活性。本试验利用昆虫-杆状病毒表达系统表达的重组H7亚型HA、HA1蛋白为可溶性的，带有6个组氨酸标签，较易纯化。重组HA、HA1蛋白具有良好的抗原性。因此，杆状病毒高效表达的重组HA、HA1蛋白在H7亚型禽流感病毒诊断及新型亚单位疫苗研究方面具有发展前景。更多还原

【Abstract】 Avian influenza(AI)is caused by type A avian influenza virus,a member of Orthomyxoviridae.Avian influenza with low virulence is characterized by respiratory symptom and lowproduction.The highly pathogenic AIV once outbreak,will cause fairly severity economic damageto China and even many worldwide poultry countires.Contrast to H5 AIV, the outbreak of H7 AIVwill also cause great damage to poultry.The H7 AIV not only make incursions into chicken, duckbut also pathopoiesia bird(H7N1),and even infect humans. The AIV genome which encodes 10proteins related to the viral structure and function is comprised of eight negative-strand RNA.,andthe hemagglutinin (HA) was the main protein of surface spikes. The hemagglutinin (HA) is avirulence-associated glycoprotein. The hemagglutinin can induce neutralizing antibodies in vivo.Therefore, the hemagglutinin plays an important role in diagnose and control avian influenza.In this study, I took pMD18-T-H7-HA conserved in our lab as template,removal nucleotidewhich encode signal tide. Using Primer5.0 and Oligo4.0,respectively design HA and HA1 twoprimers.The HA and HA1 gene were amplied by PCR from pMD18-T-H7-HA. The PCR productwas respectively inserted into SacⅠand KpnⅠMCS of the vector pBlueBachis2A.They weredesignated as pBlueBacHis-H7-HA and pBlueBacHis-H7-HA1.Purified pBlueBacHis-H7-HA andpBlueBacHis-H7-HA1 and Bacmid-N-blue DNA were cotransfected into insect cell Sf9,thenrecombinant baculovirus named P1 was harvested after 5d. P1 was on monolayer Sf9 after the thirdplaque purification to reproduce in quantity, which was named P2. After P2 was inoculated on Sf9monolayer cell 4d the CPE cells were harvested.The SDS-PAGE analysis showed that HA and HA1gene were expressed in recombinant baculovirus, molecular weight is about 66ku and 42ku.Westernblotting and Dot-ELUSA analysis proved that the recombinant protein had good reactiveability against H7 subtype serum and did not have cross reaction against chicken H5 and H9antiserum. The hemagglutinin and indirect immunofluorescence ceterified that HA and HA1 hadgood reactivity.The expressed H7 recombinant HA and HA1 protein which took use of bacilovirus systemwas soluble,6 His-tag was easily purified.The recombinant HA and HA1 had good reactivity. Theresults showed that the recombinant H7-HA and HA1 gene was specifically expressed in Sf9 cellswith the expected sizesand the expression proteins had immunoreactivity in immunoblot assay.更多还原