论文部分内容阅读
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 fragment of HBVpreC / C with a length of 640 bp was obtained by PCR and cloned into the vector pSXIVIV + X3 / 4 to construct the recombinant plasmid pSXIVVI + X3 / 4-pC / C. The recombinant strain TnNPV-pC / C-OCC + was constructed by co-transfection, which can form polyhedrosis and preC / C genes. The preC / C gene in this recombinant strain is doubly regulated by a tandem double promoter-synthesis promoter and a Hindlll linker-containing XIV promoter. Spodoptera frugiperda (Sf) cells infected with the recombinant strain and their culture supernatant were detected by solid phase radioimmunoassay with HBeAg and HBcAg. The infected cells and their culture supernatants showed HBeAg-positive and HBcAg-negative, respectively. Western analysis showed that the total protein of infected cells in 26kD and culture supernatant 15kD, showed a specific reaction with HBeAg monoclonal antibody bands. The results showed that the cloned hepatitis B virus preC / C gene was correctly expressed and processed in the baculovirus vector system, and the processed product can be effectively secreted.