中文摘要：

目的 研究靶向乙型肝炎病毒（HBV）C基因的shRNA（shRNA）诱导RNAi（RNAi）在BHK-21细胞中抑制HBV的复制与表达及抗病毒效果.方法 根据成都军区昆明总医院传染病中心克隆测序的中国56个民族CHB患者HBV基因组（基因型B属ayw1亚型）已在GenBank登录注册：CYN/2002和CYN/2000（GenBank登录号AY517488,AY517489,等）,为了监测siRNA功能提供报告基因系统,将HBV C基因PCR产物克隆构建成报告基因表达载体pC-EGFP-N1和载体pCDNA3.1B（-）,设计并构建两个靶向同源（CYN/2002和CYN/2000）毒株HBV C基因的长24核苷酸（nt）的shRNA表达质粒（S1和S2）,随机设计的用于对照的非同源长24 nt的shRNA表达质粒S3,将其克隆到载体pU6上,构建成表达目的 shRNA重组表达载体,并与pC-EGFP-N1共转染BHK-21细胞.首先在BHK-21细胞中使用BH-2型荧光显微镜观察和FACS-440型流式细胞仪检测绿色荧光蛋白（EGFP）表达细胞数量,评估该shRNA表达质粒在转染后不同时间对C基因/EGFP融合报告基因表达的抑制作用,接着在BHK-21细胞中通过实时荧光定量PCR（RT-PCR）进一步检验了shRNA抗病毒效果.结果 通过BH-2型荧光显微镜观察和FACS-440型流式细胞仪检测,发现在shRNA表达质粒和pC-EGFP-N1共转染BHK-21细胞24 h后,与pC-EGFP-N1或pEGFP-N1单质粒转染相比,S1或S2的共转染组、S1＋S2的共转染组使EGFP的表达水平降低了90%,而对照质粒S3或pU6共转染组无显著降低EGFP的表达水平,差异有统计学意义（P＜0.01） 应用RT-PCR检测C基因mRNA和EGFP基因mRNA的表达量,结果与BH-2型荧光显微镜和FACS-440型流式细胞仪检测结果相吻合,差异有统计学意义（P＜0.01）.进一步证实了RNAi抗病毒效果,抗病毒效应持续时间超过48 h.结果 发现,创建的靶向C基因的shRNA能够有效特异地抗C-EGFP基因在BHK-21细胞中的复制与表达.结论 靶向C基因的RNAi技术能够有效特异地抗HBV在BHK-21细胞中的复制与表达.RNA

英文摘要：

Objective The vaccines currently developed against infectious diseases fail to induce effective antiviral immune responses to control an abrupt outbreak of viral diseases epidemic in a short space of time.Hence there is an urgent need to develop emergency vaccines capable of producing prophylactic effects against infectious diseases.RNA interference（RNAi）is a mechanism that inhibits gene expression by causing the degradation of specific RNA molecules or hindering the transcription of specific genes.The rapidity and uniqueness of RNAi responses can make up for the current inadequacy of antiviral preventive regimes.Here we evaluate the antiviral potential of short hairpin RNA（shRNA）targeting C（core）gene of hepatitis B virus（HBV）.It plays essential roles in HBcAg encoding and viral attachment to susceptible cells during its life cycle.The present study was intended to investigate the inhibitory effect of C-specific shRNAs on HBV replication and expression in BHK-21 cells.Methods The reporter gene expression vector of pC-EGFP-N1 was constructed by cloning the DNA（PCR product）of HBV C into the EcoR Ⅰ-Hind Ⅲ sites of pEGFP-N1 to fuse C to enhanced green fluorescent protein（EGFP）for providing a reporting system for monitoring siRNA function.Plasmid pC was constructed by cloning the DNA of HBV C into the EcoR Ⅰ-Hind Ⅲ sites of pCDNA3.1B（-）directly under the control of cytomegalovirus promoter.Two plasmids（S1 ＆ S2）were constructed to express shRNAs targeting C of HBV with the length of 24 nucleotide（nt）homologous in sequence to the HBV C gene.Plasmids were designed and synthesized according to the HBV genome（HBV genotype B, ayw1 subtype）of chronic hepatic B patients from 56 ethnic minorities in China.After cloning and sequencing, the investigators registered them with the GenBank accession numbers of AY517488（CYN/2002）and AY517489（CYN/2000）, etc.Simultaneously, one of nonspecific shRNA-S3 with a length of 24 nt was also designed randomly for negative control.After