化学合成多肽体外抗HBV复制的研究

Chemically synthesized polypeptides inhibit HBV replication in vitro

摘要:

目的 观察人工设计并化学合成的多肽在体外乙型肝炎病毒（HBV）复制模型中对HBV-DNA复制、病毒标志物表达的影响，及其细胞毒性强弱，筛选高HBV抑制且低细胞毒性的多肽，探索多肽作为新型HBV抗病毒药物分子的潜力。方法 采用传统的甲基丙烯酸聚合物平台设计并合成的7种多肽（KBDT-1、2、3……7，以疏水性及阳离子电亲和力大小为依据），将7种化学合成多肽（10 mg/mL）、拉米夫定（1 mg/mL，阳性对照）、空白溶剂（阴性对照）作用于HepG 2.2.15细胞系，检测化学合成多肽对HBV的抑制作用，采用结晶紫染色法检测细胞存活率，比较各组药物对细胞毒性的强弱。选取HBV抑制作用最强的多肽，设置10、1、0.1 mg/mL浓度梯度，分别处理HepG 2.2.15细胞3、6、9 d后，收集细胞上清液，采用实时荧光定量聚合酶链反应（RT-PCR）检测病毒DNA拷贝量，化学发光微粒子免疫分析法检测HBsAg及HBeAg的变化。结果 从7种化学合成多肽中筛选出多肽KBDT-2，RT-PCR结果显示，KBDT-2具有体外抗HBV复制作用，且药物浓度越高，抑制效果越好；化学发光微粒子免疫分析法检测显示，KBDT-2对HBV生物学标志物HBsAg及HBeAg有抑制作用；结晶紫染色检测结果显示，KBDT-2对HepG 2.2.15无明显毒性作用。结论 KBDT-2具有抑制HBV复制作用，且无明显细胞毒性，可以有效降低HBsAg、HBeAg表达，为探索抗HBV新型药物提供了实验数据支持。

Abstract:

Objective To observe the effect of artificially designed and chemically synthesized polypeptides on HBV-DNA replication and expression of viral markers, as well as their cytotoxicity, screen polypeptide of high inhibition and low cytotoxicity HBV, and explore the potential of polypeptides as novel antiviral molecules for HBV. Methods Seven polypeptides (KBDT-1, 2, 3...7 based on hydrophobicity and cationic electroaffinity) were designed and synthesized using traditional methacrylic acid polymer platform, seven chemically synthesized polypeptides (10 mg/mL), lamivudine (1 mg/mL, positive control) and blank solvent (negative control) were applied to HepG 2.2.15 cell line, the inhibition effect of chemically synthesized polypeptides on HBV was detected, cell viabi-lity rate was detected by crystal violet staining, cytotoxicity of each group was compared. Polypeptide with the strongest inhibition effect on HBV was selected, concentration gradients of 10, 1 and 0.1 mg/mL were set, after 3, 6 and 9 days of treatment on HepG 2.2.15 cells, the supernatant of cells was collected, copies of viral DNA were detected by real-time fluorescence quantitative polymerase chain reaction (RT-PCR), changes in HBsAg and HBeAg were detected by chemiluminescent microparticle immunoassay. Results Peptide KBDT-2 was screened from seven chemically synthesized polypeptides, RT-PCR result showed that KBDT-2 had anti-HBV replication effect in vitro, and the higher the concentration of KBDT-2, the better the inhibition effect; chemiluminescence microparticle immunoassay showed that KBDT-2 could inhibit HBsAg and HBeAg, the biological markers of hepatitis B virus; crystal violet staining result showed that KBDT-2 had no obvious toxicity to HepG 2.2.15. Conclusion KBDT-2 can inhibit the replication of HBV without obvious cytotoxicity, and can effectively reduce the expression of HBsAg and HBeAg, which provides experimental data support for exploring new anti-HBV drugs.