表达A3C的HBV载体质粒抑制HBV的研究

Inhibitory effect of a HBV vector plasmid expressing A3C on HBV replication

目的观察表达人类载脂蛋白B mRNA编辑酶催化多肽3C(A3C)的复制缺损型HBV载体质粒抑制乙型肝炎病毒(HBV)的作用。方法利用PCR技术和基因重组方法构建表达A3C和人绿色荧光蛋白(hrGFP)的HBV载体质粒pCH-LJ3-A3C、pCH-LJ3-hrGFP,分别与野生型HBV质粒pCH-3093共转染HepG2细胞,提取细胞裂解液及细胞培养上清液中病毒颗粒DNA进行Southern blot检测;提取细胞裂解液中核心蛋白相关的HBV DNA,PCR扩增,克隆,测序。结果pCH-LJ3-A3C对细胞浆及细胞培养上清液中病毒颗粒DNA具有抑制作用,使细胞浆内HBV DNA减少31%,使上清液中子代病毒颗粒HBV DNA减少40%;对HBV DNA具有编辑作用,50个克隆测定HBV DNA序列,36克隆出现G-A突变,G-A突变总数量982位点。结论pCH-LJ3-A3C可以抑制HBV复制,pCH-LJ3-A3C对HBV DNA的编辑作用是其发挥作用的主要原因,pCH-LJ3-A3C可以作为一种新型的抗病毒制剂治疗HBV感染。

Objective To observe the inhibitory effect of a replication-defective hepatitis B virus （HBV） vector plasmid expressing A3C on HBV replication in vitro. Methods The HBV vector plasmisd pCH-LJ3-A3C and pCH-LJ3-hrGFP expressing A3C and hrGFP were constructed using PCR and gene recombination technique. The two recombinant plasmids were separately cotranfected into HepG2 cells along with the wild-type HBV plasmid pCH-3093. The HBV DNA in the cell cytoplasmic lysates and in the cell culture supematant was extracted for Southern blotting, and the nucleocapsid-associated HBV DNA were amplified by PCR, cloned and sequenced. Results pCH-LJ3-A3C showed obvious inhibitory effect on HBV DNA in the cytoplasmic lysates and cell culture supematant, causing a reduction of the HBV DNA by 31% and 40%, respectively. The pCH-LJ3-A3C plasmid was capable of editing the HBV DNA. Among the 50 sequenced clones, 36 clones had G-A mutations, with a total of 982 such mutations. Conclusion pCH-LJ3-A3C can inhibit the replication of HBV primarily by editing HBV DNA. The pCH-LJ3-A3C plasmid may serve as a new antiviral agent against human HBV infection.