摘要
通过ＰＣＲ获得长度为６４０ｂｐ的ＨＢＶｐｒｅＣ／Ｃ基因片段，将其克隆入转移载体质粒ｐＳＸＩＶＶＩ＋Ｘ３／４，构建出重组质粒ｐＳＸＩＶＶＩ＋Ｘ３／４－ｐＣ／Ｃ。利用共转染的方法，构建出既能形成多角体又能表达ｐｒｅＣ／Ｃ基因的重组毒株ＴｎＮＰＶ－ｐＣ／Ｃ－ＯＣＣ＋。该重组毒株中ｐｒｅＣ／Ｃ基因受到串联的双启动子－合成启动子和含ＨｉｎｄⅢ接头的ＸＩＶ启动子的双重调控。对感染重组毒株的草地夜蛾（Ｓｐｏｄｏｐｔｅｒａｆｒｕｇｉｐｅｒｄａ，Ｓｆ）细胞及其培养上清分别进行ＨＢｅＡｇ、ＨＢｃＡｇ固相放射免疫检测，发现受感染细胞及其培养上清均呈ＨＢｅＡｇ阳性，ＨＢｃＡｇ阴性；进一步做Ｗｅｓｔｅｒｎ分析表明，受感染细胞总蛋白中２６ｋＤ及培养上清１５ｋＤ处，呈现与ＨＢｅＡｇ单克隆抗体特异性反应条带。结果表明，克隆的乙肝病毒ｐｒｅＣ／Ｃ基因在杆状病毒载体系统中得到正确表达和加工，加工产物能够有效分泌。
The PCR-amplified HBV preC/C gene(640bp) was inserted into the transfer vector pSXIVVI +X3/4, the resultant pSXIVVI +X3/4-pC/C cotransfected Spodoptera frugiperda (Sf) cells with the parental virus(TnNPV-SVI --G) DNA to roduce the recombinant virus TnNPV-pC/C-OCC +, in which the preC/C gene was driven by the synthetic and XIV promoters. When HBeAg and HBcAg were detected by solid-phase radioimmunoassay, the cell lysates and the media from TnNPV-pC/C-OCC +-infected Sf cells were HBeAg positive and HBcAg negative. The Western blot analysis showed that the preC/C gene product, 26kD protein, was processed and secreted as 15kD protein. These results demonstrated that the preC/C gene product can be processed and secreted HBeAg efficiently in baculovirus vector system.
出处
《病毒学报》
CAS
CSCD
北大核心
1997年第4期325-331,共7页
Chinese Journal of Virology
关键词
乙型肝炎病毒
preC/C基因
杆状病毒载体
PCR, HBV preC/C gene, Baculovirus expression vector, Expression and procession, Secrete
