摘要
目的构建B和C基因型重组HBV表达载体，检测其在Huh7细胞内的DNA复制和HBsAg、HBeAg的表达。方法扩增B和C基因型HBV全基因组，并将其连接于真核表达载体pHY106，将这2个载体分别转染Huh7细胞，以pHY106空载体转染作对照。Southern印迹法检测转染72h后HBVDNA的复制，实时定量PCR检测转染后24、48、72、96和120hHuh7细胞内HBVDNA水平，ELISA检测转染后24、48、72、96和120h细胞培养上清液中HBsAg和HBeAg的表达。结果成功构建了B和C基因型HBV表达载体。转染Huh7细胞后72h，Southern印迹法检测到细胞内HBV核心颗粒内的HBV复制中间体，包括松弛环状DNA、双链DNA和单链DNA。实时定量PCR检测发现病毒DNA复制水平可达8lg拷贝／mL，ELISA结果显示HBsAg和HBeAg的表达于转染后72h达高峰，然后逐渐下降。结论成功构建B和C基因型重组HBV真核表达载体，并能在Huh7细胞内高水平复制和表达，为进一步研究H13V的结构与功能、基因表达与调控，以及抗HBV药物的筛选等提供了良好的平台。
Objective To construct recombinant full length genotype B and C hepatitis B virus （HBV） and to examine HBV DNA replication and hepatitis B surface antigen （ HBsAg）, hepatitis B e antigen （HBeAg） expressions in Huh7 cells. Methods The full length genotype B and C HBV DNA were extracted and amplified from two HBV infected patients. The recombinant plasmids were constructed by inserting the amplified HBV fragments into the eukaryotic expression vector, pHY106, which were then transfeeted into Huh7 cells. The cells transfected with blank pHY106 vector were used as control. HBV DNA replication at 72 hours of transfection was detected by Southern blot. The HBV DNA levels in Huh7 cells at 24, 48, 72, 96 and 120 hours of transfection were determined by real-time polymerase chain reaction （PCR）. Meanwhile, the HBsAg and HBeAg expression levels in the supernatants at 24, 48, 72, 96 and 120 hours were determined by enzyme linked immunosorhent assay （ELISA）. Results The recombinant plasmids expressing genotype B or C HBV DNA were successfully constructed. The HBV replicative intermediates in HBV core particles, including reDNA, dsDNA and ssDNA, were detected by Southern blot. HBV DNA level could reach 8 lg copy/mL which was by real-time PCR. HBsAg and HBeAg levels determined by ELISA peaked at 72 hours after transfection and then declined gradually. Conclusions The recombinant plasmids inserted with genotype B or C HBV DNA are constructed successfully, which can express high levels of HBsAg and HBeAg in Huh7 cells. This system provides a platform for studying the pathogenesis of B and C genotype HBV, the interaction between HBV and host, as well as exploiting new drugs against HBV.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2010年第1期10-13,共4页
Chinese Journal of Infectious Diseases
关键词
肝炎病毒
乙型
重组
遗传
DNA复制
基因表达
真核细胞
Hepatitis B virus
Recombination, genetic
DNA replication
Gene expression
Eukaryotic cells
