摘要
通过ＰＣＲ扩增，得到苜蓿丫纹夜蛾核型多角体病毒（ＡｕｔｏｇｒａｐｈａｃａｌｉｆｏｒｎｉｃａＮｕｃｌｅａｒＰｏｌｙｈｅｄｒｏｓｉｓＶｉｒｕｓ，ＡｃＮＰＶ）具早晚期启动子元件的ｐ３５基因启动子，将其插入到杆状病毒转移载体质粒ｐＳＸＩＶＶＩ＋Ｘ３多克隆位点上游，使之与ｐＳＸＩＶＶＩ＋Ｘ３质粒中的人工合成后期启动子（ＰＳｙｎ）、多角体ＸＩＶ启动子（ＰＸＩＶ）串联构成早期、晚期、极晚期能持续启动外源基因表达的转移载体质粒ｐＳＸ３５．将ｐＳＸ３５用于组建含ＨＢｓＡｇ基因并形成多角体的重组ＴｎＮＰＶ，ＨＢ－ｓＡｇ基因的表达量显著提高，表达时间亦明显提前，从而实现了外源基因在杆状病毒表达系统的全期、高效表达．ｍＲＮＡ引物延伸试验结果显示，Ｐｐ３５在重组病毒中可产生２套转录本，分别于病毒感染的早期和晚期起始ＨＢｓＡｇ基因的表达．
The promoter of p 35 gene, which is highly active at early stage of viral replication cycle and possesses both early and late regulatory elements, was fused to the reporter gene encoding hepatitis B virus surface antigen (HBsAg) and inserted into the plasmide pSXIVVI +X3 with late and very late (synthetic and XIV) promoters to yield a new transfer vector plasmid pSX35 HBs with a whole phase promoter cassette. The recombinant baculovirus designated TnNPV SX35 HBs with occlussion plus (OCC +) was also successfully constructed, which contained the HBsAg gene under the triple contral of the p 35, Syn, and XIV promoters. The expression products of HBsAg gene could be detected at 4 hours postinfection (hpi). And the expression level of HBsAg driven by the whole phasepromoter cassette was increased by 1 8 and 0 6 folds comparing with that driven by the late/very late promoters at 24 hpi and 96 hpi, respectively. These data suggest that the expression time of foreign gene was moved up, and the expression level was significantly increased by the recombinant baculovirus due to the added P p 35. The mRNA was isolated from cells at the indicated times after infection for primer extension. Six extension products of P p 35 were generated, and their initiate sequences were TTAAG and CATAGC, showing that they were the late and early initiate sites, respectively, for gene transcription.
出处
《中山大学学报（自然科学版）》
CAS
CSCD
北大核心
1997年第6期1-5,共5页
Acta Scientiarum Naturalium Universitatis Sunyatseni
基金
国家自然科学基金
广东省自然科学基金
关键词
杆状病毒载体
全期启动子盒
基因载体
基因表达
Baculoviuses vector, whole phase promoter cassette, p 35 promoter, HBsAg, mRNA primer extension
