VP22融合型显性负性突变体抑制乙肝病毒复制

目的: 了解VP22融合型显性负性(DN)突变体对抑制乙肝病毒(HBV)复制的作用. 方法: 将VP22全长及其不同区段融合于HBV核心蛋白(HBc)的C端,克隆入pcDNA3.1(-)构成DN突变体真核表达质粒. 转染HepG2.2.15细胞后,免疫荧光鉴定DN突变体在细胞内的表达,并以培养上清HBV表面抗原(HBsAg)的浓度为指标,观察DN突变体对HBV病毒复制的抑制效应. 同时以MTT比色法观察转基因的表达对宿主细胞生长状态的影响. 结果: VP22及其不同区段构建的DN突变体可在HepG2.2.15细胞中进行表达,且对宿主细胞无毒性. VP22融合型DN突变体可有效地抑制HBV的复制,具有蛋白转导特性的DN突变体比无转导特性的DN突变体具有更强的抗病毒能力. 其中VP22全长构成的DN突变体具有最强的抑制效应,可使上清HBsAg浓度下降81.94%. 结论: VP22融合型DN突变体可以增强DN突变体对HBV复制的抑制.

Inhibition of hepatitis B virus replication by dominant negative mutant of VP22 fusion protein

AIM: To investigate the inhibitory effect on hepatitis B virus (HBV) replication with dominant negative (DN) mutant of VP22 fusion protein. METHODS: Fulllength or fractions of VP22 were fused to C terminal of HBV core protein (HBc), and cloned into pcDNA3.1(-) vector, yielding eukaryotic expression plasmids of DN mutant. After transfection into HepG2.2.15 cells, the expression of DN mutant was identified by immunofluorescence staining. The inhibitory effect of DN mutant on HBV replication was indexed as the concentration of HBV surface antigen (HBsAg) in the supernatant of cell culture. And MTT assay was performed to detect the cytotoxicity of transgene expression to the host cells. RESULTS: DN mutant of VP22 and its fractions could be expressed in HepG2.2.15 cells, and had no toxic effect on the host cells. The DN mutant could inhibit HBV replication, and the mutant with protein transduction ability had a stronger inhibition than that without. The DN mutant of full-length VP22 had the strongest inhibitory effect, reducing the HBsAg concentration by 81.94%. CONCLUSION: DN mutant of VP22 fusion protein can enhance the inhibition of HBV replication.