禽流感病毒A/PFV/Restock/34(H7N1)HA基因在昆虫/杆状病毒系统中的表达  

Expression of avian influenza virus A/PFV/Restock/34(H7N1) HA gene in recombinant baculovirus

作　　者：李贺[1] 孟庆文[1] 冉多良[2] 于康震[1] 陈化兰[1] 李永强[1] 吴东来[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001 [2]新疆农业大学动物医学院,新疆乌鲁木齐830052

出　　处：《中国预防兽医学报》2006年第3期294-297,共4页Chinese Journal of Preventive Veterinary Medicine

摘　　要：经RT-PCR扩增了禽流感病毒A/PFV/Restock/1/34(H7N1)1.7kbHA基因的cDNA,将其克隆到pMD18-T中并测序。在去除编码HA信号肽的核苷酸序列后,亚克隆到杆状病毒转移载体pBlueBacHis,筛选到重组质粒命名为pBlueBacHisH7HA,测序正确后与线性化的杆状病毒DNA(Bac-N-BlueTMDNA)共转染Sf9昆虫细胞,挑取蓝色蚀斑,经三轮蚀斑纯化,获得数株重组杆状病毒rpBlueHisH7HA。提取重组病毒DNA,经PCR证明目的基因片段已插入到杆状病毒基因组中,血凝实验、SDS-PAGE和Westernblot实验结果表明HA基因在重组杆状病毒感染的HFive细胞中获得了表达。The complementary DNA of 1.7 kb HA gene was prepared from the cDNA by RT-PCR. The amplified fragment was cloned into the plasmid vector pMD18-T and then sequenced. The sequence coding of signal peptide HA was deleted, then subcloned into the downstream of the baculovirus transfer vector pBlueBacHis that contain the honeybee melittin secretion signal. The recombinant transfer vector was screened at random by picking colonies and designated pBHisH7HA. The pBHisH7HA was sequenced and co-transfected the sf9 cell with the Bat-N-BlueTM DNA by the technique of cationic liposome mediated transfection. The recombinant baculovirus was purified by three cycles of plaques assay with the chromogenic substracte X-gal in the low temperature melting agarose, and identified by PCR,HA test,SDS-PAGE and Western blot test, which proved it have good biological activity.

关 键 词：禽流感病毒 血凝素基因 重组杆状病毒 表达 

分 类 号：S852.659.5[农业科学—基础兽医学]