表达带His-tag的禽流感病毒HA基因杆状病毒转移载体的构建及重组病毒的鉴定

经RT-PCR扩增了禽流感病毒A／Goose／GuangDong／1／96（H5N1）1．7kb HA基因的eDNA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pBlueBacHis4．5，筛选到重组质粒命名为rpBacHisH5HA。测序正确后，在脂质体介导下，与线性化的杆状病毒ENA（Bac-N-Blue TMDNA）共转染Sf9昆虫细胞，挑取蓝色蚀斑，经3轮蚀斑纯化，获得数株重组杆状病毒rBacHisH5HA。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。

The Construction of Expression of His-tag Avian Influenza Virus HA Gene Baculovirus Transfer Vector and Identification of Recombinant Baculovirus

The complementary DNA of 1.7 kb HA gene was prepared from the(A/Goose/GuangDong/1/96)(H5N1) AIV 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 baculovirus transfer vector pBlueBacHis4.5. The recombinant plasmid was screened at random by picking colonies and designated rpBachisH5HA.The rpBacHisH5HA was sequenced and co-transfected the Sf9 cell with the lined Bac-N-Blue~(TM) 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.