【作者】 黄俊杰；

【导师】 成细瑶；

【作者基本信息】 广西大学 ， 轻工技术与工程， 2025， 硕士

【摘要】 严重急性呼吸系统综合征冠状病毒2（SARS-CoV-2）引发的新冠肺炎疫情（COVID-19）在过去五年间对人类社会公共卫生和经济产生了前所未有的影响而备受关注。该病毒主要通过其表面Spike蛋白（S）的受体结合域（RBD）与宿主细胞表面的血管紧张素转化酶2（ACE2）特异性识别进而感染宿主细胞。由于SARS-CoV-2存在易突变性,导致目前市面上尚无完全有效抑制该病毒感染的药物。因此,本研究拟基于上述感染机制,设计并筛选出能够靶向Spike/ACE2作用位点的多肽抑制剂,为COVID-19的临床药物研发提供潜在的候选药物。本研究的主要结果如下:（1）构建SARS-CoV-2假病毒药物筛选模型和建立细胞固相ELISA法测定药物/靶蛋白亲和力的策略。首先,通过三质粒转染系统,成功构建了基于HIV-1病毒载体的SARS-CoV-2假病毒模型。随后在Spike蛋白不同位点中引入穿膜肽TAT,实验数据显示将无规则卷曲的841～847序列替换为TAT后,构建的Spike-TAT-1筛选模型感染细胞的整体相对荧光值水平从10²提升至10⁴,最高浓度下的感染力相较野生型假病毒模型提升约50倍。基于ACE2与s RBD（spike-RBD）特异性结合的原理,建立了细胞固相ELISA方法检测ACE2配体浓度。首先,通过瞬时转染293T贴壁细胞过表达膜蛋白ACE2,ELISA测定显示s RBD与ACE2的结合平衡浓度约为10μg/m L。在此条件下,加入10μg/m L的s RBD后,待测配体可与s RBD竞争性结合ACE2,ELISA方法检测到的s RBD浓度变化直接反映了配体与ACE2的亲和力高低。（2）多肽药物设计与活性鉴定。基于Spike/ACE2复合物结构,设计了ACE2源多肽MT-03和RBD源多肽MH-1、MH-2以及MH-3,并通过订书肽策略固定MT-03的α-螺旋结构设计出MT-04。通过Spike-TAT-1假病毒筛选模型测定其抗SARS-CoV-2病毒活性。结果显示,ACE2源多肽MT-04对假病毒模型的抑制率可达92.88%,显著高于阳性对照Arbidol。细胞固相ELISA实验同样证明MT-04与s RBD亲和力最高,其IC₅₀值达到4.864μM。对MT-04的抗病毒特性进一步验证发现,使用MT-04预处理细胞（-1 h）或MT-04和SARS-CoV-2假病毒同时加入感染体系（0 h）能有效维持MT-04的中和活性,高浓度（50μM）下的MT-04能有效阻碍SARS-CoV-2 Spike介导的细胞膜融合过程且不产生细胞毒性。（3）MT-04中和突变体假病毒能力测定。通过分析SARS-CoV-2突变体毒株,构建了K417N、T478K、N501Y、D614G以及XBB.1.16突变体假病毒模型,对各突变体的感染力进行分析和对MT-04中和突变体假病毒的能力进行了验证。结果表明除了K417N突变外,上述突变导致假病毒的感染力在不同细胞中提升了28.7%～88.3%。MT-04对多个单突变体的抑制活性有所下降,其中对XBB.1.16变体的抑制活性相比野生型降低了20.75%～45.90%。而药物联用结果表明,MT-04与Arbidol合用可以补偿部分因突变导致的活性损失。更多还原

【Abstract】 The COVID-19 pandemic,caused by Severe Acute Respiratory Syndrome Coronavirus 2（SARS-CoV-2）,has garnered significant global attention over the past five years due to its unprecedented impact on public health and the global economy.This virus primarily infects host cells by binding specifically to angiotensin-converting enzyme 2（ACE2）on the host cells surface,facilitated by the receptor-binding domain（RBD）of the Spike protein（S）.Due to the high mutability of SARS-CoV-2,there are currently no fully effective drugs available to inhibit the viral infection.In light of this,the present study aims to design and identify peptide inhibitors targeting the Spike/ACE2 interaction site,offering potential drug candidates for the clinical development of COVID-19 therapeutics.The key findings of this study are as follows:（1）Develop a drug screening model for the SARS-CoV-2 pseudovirus and establish a cell-based solid-phase ELISA strategy to assess the affinity between drugs and target proteins.Initially,a SARS-CoV-2 pseudovirus model based on the HIV-1 viral vector was successfully constructed using a three-plasmid transfection system..Following this,the transmembrane peptide TAT was introduced at different sites within the Spike protein.Experimental data revealed that replacing the randomly folded sequences（residues 841-847）with TAT resulted in a significant increase in the relative fluorescence of infected cells in the Spikes-TAT-1 screening model,from10² to 10⁴,with infectivity rising approximately 50-fold compared to the pre-optimization model.Building on the specific binding interaction between ACE2 and the s RBD,a cell-based solid-phase ELISA was developed to measure ACE2 ligand concentration.ACE2 membrane protein was overexpressed in 293T adherent cells through transient transfection.ELISA results showed that the binding equilibrium concentration of s RBD and ACE2 was approximately 10μg/m L.Under this conditions,the adding of 10μg/m L s RBD allowed the test ligand to competitively bind to ACE2.The remaining s RBD concentration,detected by the ELISA,directly reflects the ligand’s affinity for ACE2.（2）Peptide Drug Design and Activity Peptide drug design and Activity evaluation.Based on the structure of the Spike/ACE2 complex,ACE2-derived peptide MT-03 and RBD-derived peptides MH-1,MH-2,and MH-3were designed.Additionally,MT-04 was developed by stabilizing theα-helical structure of MT-03 using the book-binding peptide strategy.The antiviral activity of these peptides was evaluated using the Spike-TAT-1pseudovirus screening model.The results showed that MT-04,derived from ACE2,exhibited an inhibition rate of 92.88%in the pseudovirus model,significantly outperforming the positive control,Arbidol.Cell-based solid-phase ELISA experiments further confirmed that MT-04 demonstrated the highest affinity for s RBD,with an IC₅₀ value of 4.864μM.Further analysis of MT-04’s antiviral properties revealed that pre-treating cells with MT-04（1 hour prior）or co-incubating MT-04 with SARS-CoV-2 pseudovirus（0hours）effectively maintained its neutralizing activity.At a concentration of50μM,MT-04 was able to effectively block the SARS-CoV-2 Spike-mediated cell membrane fusion process without causing cytotoxicity.（3）Determination of the pseudovirus ability of MT-04 neutralizing mutant.Mutant pseudovirus models for K417N,T478K,N501Y,D614G,and XBB.1.16 were constructed by analyzing existing SARS-CoV-2 mutant strains.The infectivity of each mutant was evaluated,and the neutralizing ability of MT-04 against these mutant pseudoviruses was tested.The results showed that,except for the K417N mutation,the other mutations led to an increase in pseudovirus infectivity by 28.7%to 88.3%across different cell types.MT-04 exhibited reduced inhibitory activity against several single mutants,with the inhibition of the XBB.1.16 variant decreasing by 20.75%to 45.90%compared to the wild-type virus.Combination therapy with Arbidol showed that co-administration of MT-04 could partially offset the loss of activity caused by these mutations.更多还原