【摘要】 <正> 自从美国科学家G.Smith等首次建立苜蓿尺蠖核型多角体病毒(AcNPV)转移载体表达系统以来,已被广泛用于外源基因的表达,成为世界上一新的具有巨大潜力的载体表达系统。为了进一步提高表达产量,降低成本,日本科学家前田进建立了家蚕核型多角体病毒(BmNPV)载体表达系统,并获得了高效表达。柞蚕是我国特产,以蛹滞育越冬,保存时间长,个体大,可工厂化生产。因此,组建柞蚕NPV转移载体,进而建立该载体表达系统,是目前利用昆虫活体为宿主进行外源基因表达较理想的昆虫杆状病毒载体表达系统。更多还原

【Abstract】 Two fragments, BamH I D and E, of Antheraea pernyi nuclear polyhe-drosis virus ( ApNPV)enome DNA containing the 5’ and 3’ ends of poly-hedrin gene of ApNPV were mapped and location of the gene on the fragments were determined by hibridization with OpNPV polyhedrin gene fragment as a probe. After mapping, the 1.4 kb and 2.6 kb fragments containing the 5’ and 3’ ends of the polyhedrin gene were cloned to pUC 19 and two plasmids of pAp DL and pAp 2.6 were constructed. For the oligonucleo-tide-directed mutagenesis, the 1.4 kb fragment was cloned to Ml3 and the sequences of polyhedrin promoter region and part of protein- coding seqen-ces were identified. According to the sequences of upstream and downstream from the start codon ATG,a 16-mer oligonucleotide primer was synthesized and the polyhedrin gene start codon ATG was converted to ATT by site-directed mutagenesis, screened by slot blotting hybridization and sequencing. The 1.4 kb mutant fragment was cloned to the plasmid of pAp 2.6 and a ApNPV transfer vector plasmid, pAp M2614 was constructed. In the construction of the plasmid,the sequences immediately upstream to the polyhedrin start codon and downstream neer ATG codon were remained,the former is important for regulation of transcription and the latter has a dramatic influence on the level of translation. At + 142bp downstream from ATG codon, there is a single BamH I site, the foreign genes can be inserted here for nonfused protein expression.更多还原