【摘要】 利用分子克隆技术对本室构建的高效鸡痘病毒表达载体 p1 1 S进行改造 ,在其人工合成禽痘病毒 ( FPV)强启动子 Ps的下游引入含 7个单一酶切位点的多克隆位点 ( MCS) ,构建了便于外源基因插入的通用性更强的高效单表达载体 p N1 1 S。然后将 FPV早晚期启动子 PE/ L 及其启动的鹅源新城疫病毒 NDV ZJ1株的 F基因一并插入 p N1 1 S中 ,使PE/ L 与 Ps反向串联 ,从而构建出 1个含 NDV ZJ1株 F基因的重组 FPV高效双表达载体 p N1 1 SEF,其 MCS可以用于插入其他外源基因。在此基础上 ,将 NDV ZJ1株的 HN基因插入 p N1 1 SEF的 Ps启动子下游的 MCS中 ,构建了共表达 NDV ZJ1株 F和 HN基因的重组 FPV双表达载体 p N1 1 SEFHN。将 p H1 1 SEFHN质粒 DNA与 FPV2 82 E4株共转染 CEF,得到共表达 NDV ZJ1株和 HN基因的重组 FPV,间接免疫荧光实验初步证明外源基因得到了较好的表达。表明所构建的通用高效 FPV表达载体有利于高效基因工程活载体疫苗的研制 ,具有广阔的应用前景更多还原

【Abstract】 A series of molecular manipulations were used to improve the highly efficient expression vector p11S constructed earlier by Zhu,et al.First,multiple cloning sites(MCS) which contained seven unique restriction enzyme sites was inserted into p11S to construct a new expression vector pN11S in which the MCS located downstream of the synthetic promoter Ps.Therefore,it would be more convenient for the insertion of foreign genes.Then,an expression cassette,which contained an early and late FPV promoter PE/L and the F gene of Newcastle disease virus(NDV) ZJ1 strain driven by PE/L,was inserted into pN11S to get a universal FPV expression vector pN11SEF in which PE/L and Ps were tandem in opposite orientation,and the MCS in pN11SEF could be used for the insertion of foreign genes other than F gene of NDV.For the application of pN11SEF,the HN gene of NDV ZJ1 strain was inserted into the MCS of pN11SEF,resulting in an FPV expression vector pN11SEFHN.After pN11SEFHN was transfected into CEF infected with FPV 282E4 strain,the recombinant FPV was selected,and it′s expression of F and HN was confirmed by indirect immunofluorescence assay.The universal and highly efficient expression vectors of FPV constructed by us will facilitate the development of genetic engineering viral vector vaccine.更多还原