从干扰素诱导蛋白MxA筛选抗乙肝病毒活性肽

黏病毒抗性蛋白A（myxovirus resistance protein A，MxA）是由干扰素诱导的具有重要抗乙肝病毒（hepatitis B virus，HBV）功能的蛋白质，我们前期工作发现，MxA主要依赖其中心互作结构域（central interactive domain，CID）与病毒直接相互作用发挥功能，但其具体的抗病毒功能区以及功能区是否具有独立的抗病毒活性仍不清楚。本研究拟鉴定MxA蛋白上的抗乙肝病毒活性肽。首先从全长MxA构建缺失突变体ΔCID和截短体CID，以HepG2-2-15细胞为病毒模型，分别转染空载质粒、MxA、ΔCID和CID，免疫荧光法检测转染效率，Western印迹法检测质粒表达，酶联免疫法测定细胞培养液中HBsAg、HBeAg的量及荧光定量PCR法测定乙肝病毒 DNA的量，评估CID段的抗乙肝病毒活性。根据CID段的晶体结构，缩短肽段长度，构建α1、α2、α3等9段肽段质粒，鉴定各段的抗乙肝病毒活性和细胞毒性（MTT法）。运用计算生物学手段--分子对接法预测MxA蛋白与病毒相互作用的模式和位点。结果显示,ΔCID、CID和9段肽段质粒的序列及表达正确，9段肽段的表达量未见显著性差异，无显著的细胞毒性。CID组和黏病毒抗性蛋白A组较对照组乙肝病毒的复制水平显著降低，CID组细胞上清中HBsAg、HBeAg及乙肝病毒 DNA的量分别减少了55.57%±8.48%、68.37%±6.24%、66.67%±6.40%，P＜0.01；MxA组的3个指标分别减少了61.63%±3.11%、70.77%±7.25%、73.73%±6.18%，P＜0.01；ΔCID组较对照组无明显变化。9段肽段中α1组较对照组HBsAg、HBeAg及乙肝病毒 DNA的量有显著下降，分别减少了48.33%±1.70%、70.67%±3.30%、68.95%±2.55%，P＜0.001，表明α1对乙肝病毒具有强抑制活性。分子对接的结果显示，384 ～ 408位氨基酸是MxA蛋白与病毒互作的关键位点，该区域落在α1肽段上，验证了α1是MxA蛋白抗乙肝病毒及与乙肝病毒相互作用中的关键区段。本研究筛选并鉴定出人干扰素诱导蛋白MxA上最有效的抗乙肝病毒活性肽α1，研究结果为抗乙肝病毒多肽类新药的研发奠定了基础。

Screening of Anti-Hepatitis B Virus Peptides from Interferon-Induced Protein MxA

Hepatitis B virus infection is a major public health problem worldwide. Developing novel and efficient anti-HBV agents is urgent. Human MxA, an interferon inducible protein, possesses significant antiviral activity against hepatitis B virus. Our previous studies have found that MxA inhibits HBV replication by directly interacting with HBV core protein HBc via its central interactive domain (CID). However, the precise anti-HBV domain in MxA and whether it can sufficiently exert anti HBV activity remain unclear. In this study, we aim to identify the most effective anti-HBV peptide from the full length MxA protein. Firstly, the truncated CID mutant and the CID-deleted mutant ΔCID were constructed and transfected into HepG2.2.15 cells. The transfection efficiency was assessed by immunofluorescence, and the expression of the plasmids was checked by Western blotting. To evaluate the anti-HBV activity, HBsAg and HBeAg in the cell culture media was determined by enzyme-linked immunosorbent assays and HBV DNA was measured by real-time quantitative PCR. Based on the structure of CID, plasmids coding nine shorter sequences within CID including α1, α2, and α3, were constructed and assessed for their individual anti-HBV activity and the toxicity to HepG2.2.15 cells by MTT tests. Furthermore, by means of computational biology：molecular docking, the mode and sites on MxA that interacting with HBV were imitated and predicated. The results showed that the sequences of the ΔCID, CID and nine peptide plasmids were correct and they were efficiently expressed in the cells. There was no significant difference in the expression levels of the nine peptide plasmids and their expression had no significant cytotoxicity. Compared to the control groups, the levels of HBsAg, HBeAg and HBV DNA were reduced by 55.57%±8.48%, 68.37%±6.24% and 66.67%±6.40% (P< 0.01) respectively in the CID groups, and reduced by 61.63%±3.11%, 70.77%±7.25% and 73.73%±6.18% (P< 0.01) respectively in the MxA groups, while no evident change was found in the ΔCID group. Among the nine peptides, α1 showed the highest anti-HBV activity. The levels of HBsAg, HBeAg and HBV DNA in the α1 groups were decreased by 48.33%±1.70%, 70.67%±3.30% and 68.95%±2.55% (P<0.001), respectively, compared to that in the control groups. Molecular docking demonstrated that the 384-408 amino acid region, which is located in the α1 domain, is essential for MxA to interact with HBV. Therefore, our study has identified that the α1 domian is crucial to the anti-HBV activity of MxA, and suggests that synthesized peptide based on this domain owns a strong anti-HBV potential. These findings are important for the development of new and more effective anti-HBV polypeptide drugs.