重组人血清白蛋白-干扰素α-2b 融合蛋白体外抗乙型肝炎病毒活性评价

摘要

OBJECTlVE To study the anti-HBV activity of prepared recombinant human serum aIbu-min-interferon α-2b fusion protein(HSA-IFNα-2b) in vitro. METHODS HepG2 ceIIs were infected with recombinant adenovirus with green fIuorescence protein and 1.6-foId HBV DNA(AdGFP-HBV). The ex-pression of HBV antigens,HBsAg and HBeAg in cuIture medium was detected by ELISA assay. The tox-icity of HSA-IFNα-2b on HepG2 ceIIs was evaIuated by mTT assay.The reIative expression of HBV RNA in ceIIs and the absoIute quantity of HBV DNA in cuIture supernatant were determined by quantitative PCR assay. The activity of HBV enhancer Ⅰ was detected by DuaI-Reporter gene assay. RESULTS HBV couId repIicate and express in HepG2 ceIIs after infection with AdGFP-HBV. The expression of HBsAg and HBeAg in cuIture serum of HepG2 ceIIs infected with AdGFP-HBV decreased by 51.32%(P﹤0.01)and 50.26%(P﹤0.01),respectiveIy,when HSA-IFNα-2b 500 kU·L-1 was added. The same concentration of HSA-IFNα-2b didn't inhibit the proIiferation of HepG2 ceIIs,but inhibited HBsAg in a concentration-dependent manner. The regression formuIa between HBsAg inhibitory rate(Y)and con-centration of HSA-IFNα-2b(X)was Y=21.11 IgX+11.91(r 2 = 0.954),IC50 = 63.76 kU·L-1 . HBV RNA in ceIIs and HBV DNA in the cuIture serum decreased by 52.83%(P﹤0.01)and 53.07%(P﹤0.01), respectiveIy,when HSA-IFNα-2b 500 kU·L-1 was added. The activity of enhancer Ⅰ decreased by 40.04%(P﹤0.01)when HSA-IFNα-2b 500 kU·L-1 was added. CONCLUSlON The ceII modeI of HBV repIication for evaIuating anti-HBV agents is successfuIIy estabIished. HSA-IFNα-2b exhibits noticeabIe anti-HBV effect invitro.%目的：检测重组人血清白蛋白-干扰素α-2b 融合蛋白（ HSA-IFNα-2b）体外抗乙型肝炎病毒（HBV）的活性。方法构建含1.6倍 HBV 基因组及绿色荧光蛋白的重组腺病毒（AdGFP-HBV），感染人肝细胞系 HepG2细胞，构建 HBV 复制细胞模型；ELISA 法检测 HBV 乙肝病毒表面抗原（HBsAg）和乙肝病毒 e 抗原（HBeAg）表达；mTT 法检测 HSA-IFNα-2b 对 HepG2细胞的毒性作用；定量 PCR 法检测细胞内 HBV RNA的相对表达以及细胞上清中 HBV DNA 绝对表达水平；双报告基因法检测 HSA-IFNα-2b 对 HBV 增强子Ⅰ活性的影响。结果 AdGFP-HBV 感染 HepG2细胞后可以复制表达 HBV。HSA-IFNα-2b 500 kU·L-1加入AdGFP-HBV感染的 HepG2细胞，使 HBsAg 表达降低51.32%（P﹤0.01），HBeAg 降低50.26%（P﹤0.01），而相同浓度的 HSA-IFNα-2b 并不抑制细胞的增殖。随 HSA-IFNα-2b 浓度增加，其对 HBsAg 表达的抑制作用逐渐升高，HSA-IFNα-2b 对 HBsAg 表达的抑制率（Y）与其浓度（X）的回归方程是 Y ＝21.11 IgX+11.91（r 2＝0.954），IC50为63.76 kU·L-1。HSA-IFNα-2b 500 kU·L-1使细胞中 HBV RNA 下降52.83%（P﹤0.01），培养上清中 HBV DNA 降低53.07%（P﹤0.01）。HSA-IFNα-2b 500 kU ·L-1使 HBV 增强子Ⅰ活性下降40.04%（P﹤0.01）。结论构建了可用于药物评价的 HBV 复制细胞模型，HSA-IFNα-2b 体外具有抗 HBV 活性。