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.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2006年第3期294-297,共4页
Chinese Journal of Preventive Veterinary Medicine
关键词
禽流感病毒
血凝素基因
重组杆状病毒
表达
avian influenza virus
HA gene
recombinant baculovirus
expression
