【摘要】 应用PCR方法扩增出 1 .8Kb的伪狂犬病毒糖蛋白gE基因 ,克隆到pUC1 1 9中形成重组质粒pRZE。经测序鉴定后再将gE基因定向亚克隆到杆状病毒转移载体pVL1 392中 ,形成重组质粒pVLgE。将pVLgE与杆状病毒线性DNA(BAC_N_BlueDNA)共转染Sf9昆虫细胞 ,经三轮蚀斑纯化 ,获得重组病毒rpVLgE。通过PCR方法鉴定证明gE基因正确插入到杆状病毒基因组中 ,直接免疫荧光试验和WesternBlot结果表明gE基因在重组杆状病毒感染的Sf9昆虫细胞中获得高效表达。表达的gE蛋白将作为伪狂犬病强毒和gE基因缺失弱毒疫苗鉴别诊断ELISA方法的抗原 ,为进一步扑灭伪狂犬病发挥重要作用更多还原

【Abstract】 The gE gene of Pseudorabies virus (PrV) was amplified by PCR and cloned into pUC119(designated as pRZE).After sequence determination,the gE gene was subcloned into a baculovirus transfer vector pVL1392(designated as pVLgE).Sf9 cells were co_transfected with the pVLgE and linearized Bac_N_Blue DNA,and the recombinant baculovirus harboring gE gene was identified by PCR and plaque purified three times.The expression of gE gene in Sf9 cells was determined by indirect immunofluorescent assay and western blot with polyclonal antibodies against PrV.The expressed gE protein will be used to establish a gE_ELISA for differentiation of PrV infection from vaccination and play an important role in future eradication program.更多还原