Guo Chun-xia, He Yong-wen, Peng Cheng, Lei Yan-chang, Li Wen-ting
Department of Infectious Disease, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Zhonghua Gan Zang Bing Za Zhi. 2010 Feb;18(2):105-8. doi: 10.3760/cma.j.issn.1007-3418.2010.02.007.
To investigate the effects of different PAP domains on hepatitis B virus replication.
The full length and two truncated PAP mutants were cloned into a eukaryotic expression plasmid, and were transfected into HepG2.2.15 cells using lipofectamine 2000. 3 days after transfection, the medium and cells were collected. HBsAg and HBeAg were measured using ELISA. The titers of HBV DNA were quantified using fluorogenic quantitative PCR (FQ-PCR). HepG2 cells were used to determine the cytotoxicity of the plasmids transfection by MTT assays.
The inhibitory effect on HBV replication of the C-terminal 25 amino acids deleted PAP mutant (pXF3H-PAP14) was not significantly different from that of the full length PAP (pXF3H-PAP12) (Chi-square test = 0.5, 2.0, 0.02, probability value more than 0.05), however, the cytotoxicity of pXF3H-PAP14 was lower than that of pXF3H-PAP12 (Chi-square test = 7.7, probability value less than 0.01). Both N-terminal 69 amino acids deleted mutant and C-terminal 25 amino acids deleted mutant had no cytotoxicity and no antiviral activity.
C-terminal 25 amino acid of PAP is related to cytotoxicity but not related to antiviral activity of PAP. N-terminal 69 amino acid of PAP is related to the anti-HBV effect of PAP.
研究不同聚（A）聚合酶（PAP）结构域对乙型肝炎病毒复制的影响。
将全长及两个截短的PAP突变体克隆至真核表达质粒中，使用脂质体2000转染至HepG2.2.15细胞。转染3天后，收集培养基和细胞。采用酶联免疫吸附测定法（ELISA）检测乙肝表面抗原（HBsAg）和乙肝e抗原（HBeAg）。采用荧光定量聚合酶链反应（FQ-PCR）对乙肝病毒脱氧核糖核酸（HBV DNA）滴度进行定量分析。采用噻唑蓝（MTT）法，用HepG2细胞测定质粒转染的细胞毒性。
C末端缺失25个氨基酸的PAP突变体（pXF3H-PAP14）对HBV复制的抑制作用与全长PAP（pXF3H-PAP12）相比，差异无统计学意义（卡方检验=0.5、2.0、0.02，概率值大于0.05），然而，pXF3H-PAP14的细胞毒性低于pXF3H-PAP12（卡方检验=7.7，概率值小于0.01）。N末端缺失69个氨基酸的突变体和C末端缺失25个氨基酸的突变体均无细胞毒性及抗病毒活性。
PAP的C末端25个氨基酸与PAP的细胞毒性有关，但与抗病毒活性无关。PAP的N末端69个氨基酸与PAP的抗乙肝病毒作用有关。
