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当前位置: 首页 > 文章 > 表达H7亚型禽流感HA基因杆状病毒转移载体的构建及重组病毒的鉴定 中国动物检疫 2009 (4) 32-34
Position: Home > Articles > The Construction of Expression of Avian influenza virus HA Gene Baculovirus transfer vector and Identification of Recombinant Baculovirus China Animal Health Inspection 2009 (4) 32-34

表达H7亚型禽流感HA基因杆状病毒转移载体的构建及重组病毒的鉴定

作  者：
李贺;马保华;孟庆文
单  位：
南海出入境检验检疫局;中国农业科学院哈尔滨兽医研究所
关键词：
禽流感;HA基因;杆状病毒
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pBlueBacHis4.5,筛选到重组质粒命名为rpBacHisH7HA。测序正确后,在脂质体介导下,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rBacHisH7HA。提取重组病毒DNA经PCR证明目的基因片段已插入杆状病毒基因组。
译  名：
The Construction of Expression of Avian influenza virus HA Gene Baculovirus transfer vector and Identification of Recombinant Baculovirus
作  者：
LI He1,2,MA Bao-hua2, MENG Qing-wen1 (1. Harbin Veterinary Research InstituteCAAS,Harbin,150001,China; 2.Nanhai Entry-Exit Inspection Quarantine Bureau,Guangdong ,528200,China)
关键词：
avian influenza virus;HA gene;Baculovirus
摘  要：
The complementary DNA of 1.7 kb HA gene was prepared from the A/PFV/Restock/1/34 (H7N1) 1.7kb HA AIV amplified by RT-PCR fragment,cloned into the plasmid vector pMD18-T and then sequenced. The sequence coding of signal peptide HA was deleted,then subcloned into the baculovirus transfer vector pBlue-BacHis4.5. The recombinant plasmid was screened at random by picking colonies and designated rpBachisH7HA. The rpBacHisH7HA was sequenced and co-transfected the sf9 cell with the lined Bac-N-BlueTM DNA by the technique of cationic liposome mediated transfection. The recombinant baculovirus was purified by three cycles of plaque assay with the chromogenic substracte X-gal in the low temperature melting agarose overlay and analyzed by PCR.

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