shRNA表达载体构建及其对HBV体外抑制作用

Construction of shRNA expression plasmid and its inhibitive effect on HBV replication in vitro

目的构建乙型肝炎病毒(HBV)基因X区和P区的shRNA表达载体,通过纳米聚合物将其转入HBV感染的细胞模型,并检测其对HBV复制的抑制作用。方法设计3条针对HBV基因编码区的干扰RNA序列,合成3对64nt的寡核苷酸,插入载体pRNAT-U6,构建重组干扰质粒Sl、S2和S3,利用纳米转染剂转入HBV感染的HepG2细胞。用实时荧光定量法检测HBVmRNA含量。结果干扰质粒S1、S2对HBv的复制有明显抑制作用,荧光定量检测转染后HBVmRNA表达水平发现,HBVmRNA含量分别减少至空白对照组的70.2%和61.2%,无关对照质粒则无抑制作用。结论成功构建针对HBv编码区的shRNA表达载体,并能在体外有效抑制HBVmRNA。

Objective To construct small hair RNA（shRNA） expressiion vector specifically targeting the X and P gene sequence of hepatitis B virus（HBA） and transfecting the vector into infection cell model by nanometer polymers,and to evaluate its inhibitory effcet on the replication of HBV.Methods The interfering RNA sequences targeting HBV genetic coding region were designed and three pairs of oligonucleotide（64 nt） were synthesized.Then they were inserted into pRNAT-U6 to construct recombinant interfering plasmids S1,S2 and S3.The three recombinant plasmids were transfected into HBV infected HepG2 cells by nanometer transfection reagent.HBV mRNA was measured with real time PCR.Results The replication of HBV was suppressed by the inter-fering plasmids S1 and S2 sigificantly.Compared to blank control,HBV mRNA of groups S1 and S2 were reduced to 70.2% and 61.2%,respectively（P 0.05）.Unrelated control plasmid had no inhibitory effect.Conclusion The shRNA expression vectors targeting HBV genetic coding region were successfully constructed and the study results show that they can suppress HBV mRNA in vitro.