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

The Construction of Expression of Avian influenza virus HA Gene Baculovirus transfer vector and Identification of Recombinant Baculovirus

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。

The complementary DNA of 1.7 kb HA gene was prepared from the A/PFV/Restock/1/34 （H7N1） 1.7kb HA A IV 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 pBlueBacHis4.5. The recombinant plasmid was screened at random by picking colonies and designated rpBachisH7HA. The rpBacHisH7HA was sequenced and co-transfected the st9 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.