Using pseudorabies virus Ea strain as material,we inserted LacZ gene expression cassette into gE gene.After blue plaque and plaque purification,a recombinant virus PRVEa TK+-/gE+-/LacZ++ generated.Utilizing EcoR I sit...Using pseudorabies virus Ea strain as material,we inserted LacZ gene expression cassette into gE gene.After blue plaque and plaque purification,a recombinant virus PRVEa TK+-/gE+-/LacZ++ generated.Utilizing EcoR I site in LacZ gene, digested PRVEa TK+-/gE+-/LacZ++ genome DNA was cotransfected into PK-15 cells with plasmid pFBBS,then PRVEa TK+-/gE+-/gp63+- generated after plaque purification.Four pairs of primers amplification demonstrated the virus was pure TK+-/gE+-/gp63+- mutant virus.PCR product sequence indicates there were 205bp deletion in TK gene;1247bp deletion in gE,gp63 and intergenic region of PRVEa TK+-/gE+-/gp63+- mutant virus genome DNA.Inoculation to Balb/C mice with PRVEa TK+-/gE+-/gp63+- indicates the virulence is reduced greatly.展开更多
构建了 2个利用人类巨细胞病毒 ( HCMV)的启动子启动表达伪狂犬病病毒 Ea株糖蛋白 g D基因的真核表达质粒 p CIDI和 pc DDI,体外转染 BHK-2 1细胞 ,用间接免疫荧光法检测 ,证实糖蛋白 g D在细胞中得到表达。用表达质粒 p CIDI和 pc DDI...构建了 2个利用人类巨细胞病毒 ( HCMV)的启动子启动表达伪狂犬病病毒 Ea株糖蛋白 g D基因的真核表达质粒 p CIDI和 pc DDI,体外转染 BHK-2 1细胞 ,用间接免疫荧光法检测 ,证实糖蛋白 g D在细胞中得到表达。用表达质粒 p CIDI和 pc DDI作为核酸疫苗免疫 BALB/c小鼠 ,ELISA检测小鼠血清中抗伪狂犬病病毒的抗体 ,结果其滴度为 1∶ 1 2 8～ 1∶ 51 2。初步证实 ,用 g D基因作为核酸疫苗免疫动物 。展开更多
文摘Using pseudorabies virus Ea strain as material,we inserted LacZ gene expression cassette into gE gene.After blue plaque and plaque purification,a recombinant virus PRVEa TK+-/gE+-/LacZ++ generated.Utilizing EcoR I site in LacZ gene, digested PRVEa TK+-/gE+-/LacZ++ genome DNA was cotransfected into PK-15 cells with plasmid pFBBS,then PRVEa TK+-/gE+-/gp63+- generated after plaque purification.Four pairs of primers amplification demonstrated the virus was pure TK+-/gE+-/gp63+- mutant virus.PCR product sequence indicates there were 205bp deletion in TK gene;1247bp deletion in gE,gp63 and intergenic region of PRVEa TK+-/gE+-/gp63+- mutant virus genome DNA.Inoculation to Balb/C mice with PRVEa TK+-/gE+-/gp63+- indicates the virulence is reduced greatly.
文摘构建了 2个利用人类巨细胞病毒 ( HCMV)的启动子启动表达伪狂犬病病毒 Ea株糖蛋白 g D基因的真核表达质粒 p CIDI和 pc DDI,体外转染 BHK-2 1细胞 ,用间接免疫荧光法检测 ,证实糖蛋白 g D在细胞中得到表达。用表达质粒 p CIDI和 pc DDI作为核酸疫苗免疫 BALB/c小鼠 ,ELISA检测小鼠血清中抗伪狂犬病病毒的抗体 ,结果其滴度为 1∶ 1 2 8～ 1∶ 51 2。初步证实 ,用 g D基因作为核酸疫苗免疫动物 。
