商陆抗病毒蛋白不同结构域的抗乙型肝炎病毒活性

目的 比较全长和不同片段缺失型商陆抗病毒蛋白(PAP)基因真核表达质粒体外抗HBV作用及其细胞毒性作用.方法 将全长和不同片段缺失型PAP基因真核表达质粒用脂质体转染HepG2.2.15细胞,收获生长良好的HepG2.2.15细胞,转染前1 d,接种于24孔培养细胞板,培养20 h后,待细胞密度达到40%～50%时进行转染.细胞随机分为4组:pXF3H组,转染空质粒pXF3H作为对照;pXF3H-PAP_(12)组,转染全长PAP的真核表达质粒pXF3H-PAP_(12);pXF3H-PAP_(14)组,转染C端缺失25个氨基酸的PAP的真核表达质粒pXF3H-PAP_(14);pXF3H-PAP3_(34)组,转染既缺失N端69个氨基酸又缺失C端25个氨基酸的PAP的真核表达质粒pXF3H-PAP_(34).转染的质粒剂量为每孔1.0μg,终浓度为2.0μg/ml,质粒DNA(μg)和脂质体(μl)的比例为1:2.5,转染72 h后收集细胞及培养上清液.酶联免疫吸附法检测培养上清液HBsAg和HBeAg,荧光定量PCR检测HBV DNA水平,四甲基偶氮唑盐比色法检测各质粒对转染细胞的毒性作用.应用SPSS12.0软件包处理数据,两样本均数的比较采用t检验,率的比较采用χ~2检验.结果 对HBsAg、HBeAg、HBVDNA的抑制率,pXF3H-PAP_(14)组分别为56.3%、75.8%和61.7%,pXF3H-PAP_(12)组分别为61.4%、84.2%和63.2%,两组间差异无统计学意义.但pXF3H-PAP_(14)组细胞毒性 (抑制率为10.2%)明显低于pXF3H-PAP_(12)组(抑制率为27.1%),χ~2=7.7,P<0.01.pXF3H-PAP_(34)组无细胞毒性,但其抗HBV作用也丧失,对HBsAg、HBeAg、HBV DNA的抑制率分别为7.8%、11.0%、20.5%.结论 PAP的C端25个氨基酸与细胞毒性相关,与抗HBV活性无关;PAP的N端69个氨基酸与抗HBV活性相关.

The effects of different PAP domains on hepatitis B virus replication

Objective To investigate the effects of different PAP domains on hepatitis B virus replication. Methods The full length and two truncated PAP mutants were cloned into a eukaryotic expres-sion 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. Results The inhibitory effect on HBV replication of the C-terminal 25 amino acids deleted PAP mutant (pXF3H-PAP_(14)) was not significantly different from that of the full length PAP (pXF3H-PAP_(12)) ( χ~2 = 0.5, 2.0, 0.02, P > 0.05), however, the cytotoxicity of pXF3H-PAP_(14) was lower than that of pXF3H-PAP_(12) (χ~2 = 7.7, P < 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. Conclu-sion 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.