HMGN2分子体外抗乙型肝炎病毒活性研究

Inhibitory effect of HMGN2 protein on human hepatitis B viral HBeAg and HBsAg expression and DNA replication in HBV-transformed HepG2. 2.15 cell line

目的:我们先前的研究证明HMGN2(high mobility group nucleosomal-binding domain2)是人LAK细胞抗菌的一个新的效应分子,本研究欲检测其体外抗乙型肝炎病毒的活性。方法:应用反向高效液相色谱技术从人淋巴结组织酸溶性提取物中分离纯化批量HMGN2分子,用质谱精确分子量测定、Western blotting和抗菌试验对分离纯化得到的HMGN2分子进行分子鉴定。采用MTT法检测HMGN2分子对稳定转染复制型HBV重组质粒的肝胚瘤细胞株HepG2.2.15细胞的细胞毒性;用不同浓度HMGN2分子作用于HepG2.2.15细胞,分别在第3d和第6d收集细胞培养上清液,ELISA检测上清中HBV表面抗原(HBsAg)及e抗原(HBeAg),采用实时荧光定量PCR法检测上清液HBVDNA的含量。结果:从人淋巴结组织中分离纯化获得纯度较高的HMGN2蛋白;在检1-100mg/L范围内,HMGN2分子对HepG2.2.15细胞无细胞毒性;HMGN2分子在1-5mg/L水平即可显著抑制HBeAg和HBsAg的表达,可显著降低HBVDNA拷贝数。结论:HMGN2分子在体外具有较强的抗HBV活性。

AIM： We previously demonstrated that HMGN2 is an antibacterial effector molecule in human LAK cells. This study was to examine the antiviral activity of HMGN2 against human hepatitis virus. METHODS： The purification and identify of HMGN2 proteins including preparative acid - urea polyacymide gel electrophoresis elutian, reverse - phase high - performance liquid chromatography, mass spectrum, Western blotting and antimicrobial assay were conducted. The cellular toxicity of HMGN2 to the HepG2. 2. 15 cells was detected by MTY assay. HBeAg and HBsAg expressions were measured by ELISA. HBV DNA copies were determined by real time quantitative PCR. RESULTS ： A bulk of HMGN2 was isolated and purified from the acid soluble proteins of human lymph node, and identified. The HBV - transfected HepG2. 2. 15 cell line was used in the in vitro assay system. In the range of testing 1 - 100 mg/L of HMGN2, no eytotoxicity to HepG2. 2. 15 cells was detected by MTT assay. When incubated with HMGN2 at 1 -5 mg/L for72 h or 144 h, a sig- nificant reduction in HBeAg and HBsAg expression and in HBV DNA copies was observed in the supernatant of HepG2. 2. 15 cells. CONCLUSION： HMGN2 protein markedly inhibits HBV expression and replication in vitro.