A protein-protein interaction map reveals that the Coxiella burnetii effector CirB inhibits host proteasome activity.

Coxiella burnetii is the etiological agent of the zoonotic disease Q fever, which is featured by its ability to replicate in acid vacuoles resembling the lysosomal network. One key virulence determinant of C. burnetii is the Dot/Icm system that transfers more than 150 effector proteins into host cells. These effectors function to construct the lysosome-like compartment permissive for bacterial replication, but the functions of most of these effectors remain elusive. In this study, we used an affinity tag purification mass spectrometry (AP-MS) approach to generate a C. burnetii-human protein-protein interaction (PPI) map involving 53 C. burnetii effectors and 3480 host proteins. This PPI map revealed that the C. burnetii effector CBU0425 (designated CirB) interacts with most subunits of the 20S core proteasome. We found that ectopically expressed CirB inhibits hydrolytic activity of the proteasome. In addition, overexpression of CirB in C. burnetii caused dramatic inhibition of proteasome activity in host cells, while knocking down CirB expression alleviated such inhibitory effects. Moreover, we showed that a region of CirB that spans residues 91-120 binds to the proteasome subunit PSMB5 (beta 5). Finally, PSMB5 knockdown promotes C. burnetii virulence, highlighting the importance of proteasome activity modulation during the course of C. burnetii infection.

Lipid Giant Vesicles Engulf Living Bacteria Triggered by Minor Enhancement in Membrane Fluidity.

Antibacterial amphiphiles normally kill bacteria by destroying the bacterial membrane. Whether and how antibacterial amphiphiles alter normal cell membrane and lead to subsequent effects on pathogen invasion into cells have been scarcely promulgated. Herein, by taking four antibacterial gemini amphiphiles with different spacer groups to modulate cell-mimic phospholipid giant unilamellar vesicles (GUVs), bacteria adhesion on the modified GUVs surface and bacteria engulfment process by the GUVs are clearly captured by confocal laser scanning microscopy. Further characterization shows that the enhanced cationic surface charge of GUVs by the amphiphiles determines the bacteria adhesion amount, while the involvement of amphiphile in GUVs results in looser molecular arrangement and concomitant higher fluidity in the bilayer membranes, facilitating the bacteria intruding into GUVs. This study sheds new light on the effect of amphiphiles on membrane bilayer and the concurrent effect on pathogen invasion into cell mimics and broadens the nonprotein-mediated endocytosis pathway for live bacteria.

A Strong Supramolecular Mechanophore with Controlled Mechanical Strength.

Supramolecular mechanophores typically exhibit much lower mechanical strengths than covalent counterparts, with strengths usually around 100 pN, which is significantly lower than the nN-scale strength of covalent bonds. Inspired by the slow dissociation kinetics of the cucurbit[7]uril (CB[7])-hexanoate-isoquinoline (HIQ) complex, we discovered that charge-dipole repulsion can be utilized to create strong supramolecular mechanophores. When activated at its -COO- state, the CB[7]-HIQ complex exhibits a high mechanical strength of ~700 pN, comparable to weak covalent bonds such as Au-S bonds or a thiol-maleimide adducts. The strength of the CB[7]-HIQ complex can also be tuned with pH in a gradual manner, with a minimum value of ~150 pN at its -COOH state, similar to an ordinary supramolecular conjugate. This research may pave the way for the development of supramolecular architectures that combine the advantages of covalent and supramolecular systems.

Computational design of ultrashort peptide inhibitors of the receptor-binding domain of the SARS-CoV-2 S protein.

Targeting the interaction between severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2)-receptor-binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) is believed to be an effective strategy for drug design to inhibit the infection of SARS-CoV-2. Herein, several ultrashort peptidase inhibitors against the RBD-ACE2 interaction were obtained by a computer-aided approach based on the RBD-binding residues on the protease domain (PD) of ACE2. The designed peptides were tested on a model coronavirus GX_P2V, which has 92.2 and 86% amino acid identity to the SARS-CoV-2 spike protein and RBD, respectively. Molecular dynamics simulations and binding free energy analysis predicted a potential binding pocket on the RBD of the spike protein, and this was confirmed by the specifically designed peptides SI5α and SI5α-b. They have only seven residues, showing potent antiviral activity and low cytotoxicity. Enzyme-linked immunosorbent assay result also confirmed their inhibitory ability against the RBD-ACE2 interaction. The ultrashort peptides are promising precursor molecules for the drug development of Corona Virus Disease 2019, and the novel binding pocket on the RBD may be helpful for the design of RBD inhibitors or antibodies against SARS-CoV-2.

Bovine lactoferrin inhibits SARS-CoV-2 and SARS-CoV-1 by targeting the RdRp complex and alleviates viral infection in the hamster model.

Breast milk has been found to inhibit coronavirus infection, while the key components and mechanisms are unknown. We aimed to determine the components that contribute to the antiviral effects of breastmilk and explore their potential mechanism. Lactoferrin (Lf) and milk fat globule membrane inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-related coronavirus GX_P2V and transcription- and replication-competent SARS-CoV-2 virus-like particles in vitro and block viral entry into cells. We confirmed that bovine Lf (bLf) blocked the binding between human angiotensin-converting enzyme 2 and SARS-CoV-2 spike protein by combining receptor-binding domain (RBD). Importantly, bLf inhibited RNA-dependent RNA polymerase (RdRp) activity of both SARS-CoV-2 and SARS-CoV in vitro in the nanomolar range. So far, no biological macromolecules have been reported to inhibit coronavirus RdRp. Our result indicated that bLf plays a major role in inhibiting viral replication. bLf treatment reduced viral load in lungs and tracheae and alleviated pathological damage. Our study provides evidence that bLf prevents SARS-CoV-2 infection by combining SARS-CoV-2 spike protein RBD and inhibiting coronaviruses' RdRp activity, and may be a promising candidate for the treatment of coronavirus disease 2019.

64Cu-labeled daratumumab F(ab')2 fragment enables early visualization of CD38-positive lymphoma.

PURPOSE: Abnormal CD38 expression in some hematologic malignancies, including lymphoma, has made it a biomarker for targeted therapies. Daratumumab (Dara) is the first FDA-approved CD38-specific monoclonal antibody, enabling successfully immunoPET imaging over the past years. Radiolabeled Dara however has a long blood circulation and delayed tumor uptake which can limit its applications. The focus of this study is to develop 64Cu-labeled Dara-F(ab')2 for the visualization of CD38 in lymphoma models. METHODS: F(ab')2 fragment was prepared from Dara using an IdeS enzyme and purified with Protein A beads. Western blotting, flow cytometry, and surface plasmon resonance (SPR) were performed for in vitro assay. Probes were labeled with 64Cu after the chelation of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). Small animal PET imaging and quantitative analysis were performed after injection of 64Cu-labeled Dara-F(ab')2, IgG-F(ab')2, and Dara for evaluation in lymphoma models. RESULTS: Flow cytometry and SPR assay proved the specific binding ability of Dara-F(ab')2 and NOTA-Dara-F(ab')2 in vitro. Radiolabeling yield of [64Cu]Cu-NOTA-Dara-F(ab')2 was over 90% and with a specific activity of 4.0 ± 0.6 × 103 MBq/µmol (n = 5). PET imaging showed [64Cu]Cu-NOTA-Dara-F(ab')2 had a rapid and high tumor uptake as early as 2 h (6.9 ± 1.2%ID/g) and peaked (9.5 ± 0.7%ID/g) at 12 h, whereas [64Cu]Cu-NOTA-Dara reached its tumor uptake peaked at 48 h (8.3 ± 1.4%ID/g, n = 4). In comparison, IgG-F(ab')2 and HBL-1 control groups found no noticeable tumor uptake. [64Cu]Cu-NOTA-Dara-F(ab')2 had significantly lower uptake in blood pool, bone, and muscle than [64Cu]Cu-NOTA-Dara and its tumor-to-blood and tumor-to-muscle ratios were significantly higher than controls. CONCLUSIONS: [64Cu]Cu-NOTA-Dara-F(ab')2 showed a rapid and high tumor uptake in CD38-positive lymphoma models with favorable imaging contrast, showing its promise as a potential PET imaging agent for future clinical applications.

Effect and mechanism of downregulating the long-chain noncoding RNA TM4SF1-AS1 on the proliferation, apoptosis and invasion of gastric cancer cells.

BACKGROUND: To investigate long-chain noncoding TM4SF1-AS1 in gastric cancer (GC) tissues and cells. METHODS: TM4SF1-AS1 in 40 GC tissues and adjacent tissues was detected and compared using real-time fluorescence quantitative PCR (qRT-PCR). TM4SF1-AS1 in MKN28 and SGC7901 GC cells was downregulated using small interfering RNA (shRNA). The cells were grouped into an interference group (shTM4SF1-AS1 group) and a control group (shControl group). MTT and Transwell tests were applied to determine the proliferation and invasion of the cells in both groups, and flow cytometry was performed to assess the apoptosis rate in the two groups. Western blotting was performed to determine changes in key proteins in cells during the epithelial-to-mesenchymal transition (EMT) and in the TM4SF1 and PI3K-AKT signalling pathways in response to the downregulation of TM4SF1-AS1. RESULTS: The proliferation of MKN28 and SGC7901 in the shTM4SF1-AS1 group was significantly inhibited at 48 h and 72 h compared to that in the shControl group (all P < 0.05). In the shTM4SF1-AS1 group, the number of invaded MKN28 and SGC7901 cells was significantly lower than that in the shControl group (all P < 0.05). Apoptosis in the MKN28 and SGC7901 shTM4SF1-AS1 groups was significantly higher than that in the shControl group (all P < 0.05). Compared to those in the shControl group, levels of E-cadherin in EMT-related proteins were significantly elevated (P < 0.01), while levels of N-cadherin, Snail and Twist1 were significantly decreased (all P < 0.01). After silencing the expression of LncTM4SF1-AS1, the expression levels of TM4SF1 in the shTM4SF1-AS1 group were downregulated compared to those in the shControl group, and the p-PI3K and p-AKT proteins in the PI3K-AKT signalling pathway in the shTM4SF1-AS1 group were downregulated compared to those of the shControl group. CONCLUSIONS: TM4SF1-AS1 is upregulated in gastric cancer tissues and cells. Interfering with and downregulating its expression inhibit cancer cell proliferation, invasion and the EMT and promote apoptosis. The underlying mechanism for these effects is related to silencing the TM4SF1 and PI3K-AKT signalling pathways. TM4SF1-AS1 may be a potential therapeutic target for gastric cancer.

Real-time identification of the singleness of a trapped bead in optical tweezers.

Beads trapped in optical tweezers are aligned along the optical propagation direction, which makes it difficult to determine the number of beads with bright-field microscopy. This problem also dramatically influences the measurement of the optical trapping based single-molecule force spectroscopy. Here, we propose a video processing approach to count the number of trapped micro-objects in real time. The approach uses a normalized cross-correlation algorithm and image enhancement techniques to amplify a slight change of the image induced by the entry of an exotic object. As tested, this method introduces a ∼10% change per bead to the image similarity, and up to four beads, one-by-one falling into the trap, are identified. Moreover, the feasibility of the above analysis in a moving trap is investigated. A movement of the trap leads to a fluctuation of less than 2% for the similarity signal and can be ignored in most cases. The experimental results prove that image similarity measurement is a sensitive way to monitor the interruption, which is very useful, especially during experiments. In addition, the approach is easy to apply to an existing optical tweezers system.

Staphylokinase Displays Surprisingly Low Mechanical Stability.

Single-molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulations have revealed that shear topology is an important structural feature for mechanically stable proteins. Proteins containing a ß-grasp fold display the typical shear topology and are generally of significant mechanical stability. In an effort to experimentally identify mechanically strong proteins using single-molecule atomic force microscopy, we found that staphylokinase (SAK), which has a typical ß-grasp fold and was predicted to be mechanically stable in coarse-grained MD simulations, displays surprisingly low mechanical stability. At a pulling speed of 400 nm/s, SAK unfolds at ∼60 pN, making it the mechanically weakest protein among the ß-grasp fold proteins that have been characterized experimentally. In contrast, its structural homologous protein streptokinase ß domain displays significant mechanical stability under the same experimental condition. Our results showed that the large malleability of native-state SAK is largely responsible for its low mechanical stability. The molecular origin of this large malleability of SAK remains unknown. Our results reveal a hidden complexity in protein mechanics and call for a detailed investigation into the molecular determinants of the protein mechanical malleability.

Single-Molecule Force Spectroscopy Trajectories of a Single Protein and Its Polyproteins Are Equivalent: A Direct Experimental Validation Based on A Small Protein NuG2.

Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2)4 as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2)4 lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments.

Elevated expression of CCAT2 is associated with poor prognosis in esophageal squamous cell carcinoma.

BACKGROUND AND OBJECTIVES: CCAT2, a novel long non-coding RNAs (lncRNAs), is found to promote the metastasis and invasion of colon, lung, and breast cancers. This study aimed to investigate the level of CCAT2 in esophageal squamous cell carcinoma (ESCC) and to elucidate its clinical significance. METHODS: The expression level of CCAT2 and the status of MYC amplification were examined in 229 ESCC samples using quantitative real- time PCR. RESULTS: CCAT2 was upregulated in ESCC tissues, especially in cases with lymph node metastasis (LNM), advanced TNM stages, and MYC amplification. Furthermore, the level of CCAT2 was positively correlated with TNM stages, LNM, and the number of positive lymph nodes. High CCAT2 expression and MYC amplification were significantly associated with TNM stages and LNM. Survival analyses revealed that high CCAT2 expression and MYC amplification were significantly associated with poorer overall survival in ESCC patients. Furthermore, patients with high CCAT2 expression and MYC amplification had a 2.199-fold increased risk of death compared with those with low CCAT2 expression and MYC non-amplification. CONCLUSIONS: Our study provides the first evidence associating CCAT2 expression and poor survival in ESCC. CCAT2 may be a prognostic biomarker and therapeutic target for ESCC.

Direct Observation of the Reversible Two-State Unfolding and Refolding of an α/ß Protein by Single-Molecule Atomic Force Microscopy.

Directly observing protein folding in real time using atomic force microscopy (AFM) is challenging. Here the use of AFM to directly monitor the folding of an α/ß protein, NuG2, by using low-drift AFM cantilevers is demonstrated. At slow pulling speeds (<50 nm s(-1)), the refolding of NuG2 can be clearly observed. Lowering the pulling speed reduces the difference between the unfolding and refolding forces, bringing the non-equilibrium unfolding-refolding reactions towards equilibrium. At very low pulling speeds (ca. 2 nm s(-1)), unfolding and refolding were observed to occur in near equilibrium. Based on the Crooks fluctuation theorem, we then measured the equilibrium free energy change between folded and unfolded states of NuG2. The improved long-term stability of AFM achieved using gold-free cantilevers allows folding-unfolding reactions of α/ß proteins to be directly monitored near equilibrium, opening the avenue towards probing the folding reactions of other mechanically important α/ß and all-ß elastomeric proteins.

Mechanically tightening a protein slipknot into a trefoil knot.

The knotted/slipknotted polypeptide chain is one of the most surprising topological features found in certain proteins. Understanding how knotted/slipknotted proteins overcome the topological difficulty during the folding process has become a challenging problem. By stretching a knotted/slipknotted protein, it is possible to untie or tighten a knotted polypeptide and even convert a slipknot to a true knot. Here, we use single molecule force spectroscopy as well as steered molecular dynamics (SMD) simulations to investigate how the slipknotted protein AFV3-109 is transformed into a tightened trefoil knot by applied pulling force. Our results show that by pulling the N-terminus and the threaded loop of AFV3-109, the protein can be unfolded via multiple pathways and the slipknot can be transformed into a tightened trefoil knot involving ∼13 amino acid residues as the polypeptide chain is apparently shortened by ∼4.7 nm. The SMD simulation results are largely consistent with our experimental findings, providing a plausible and detailed molecular mechanism of mechanical unfolding and knot tightening of AFV3-109. These simulations reveal that interactions between shearing ß-strands on the threaded and knotting loops provide high mechanical resistance during mechanical unfolding.

An Ultra-Conductive and Patternable 40 nm-Thick Polymer Film for Reliable Emotion Recognition.

Understanding psychology is an important task in modern society which helps predict human behavior and provide feedback accordingly. Monitoring of weak psychological and emotional changes requires bioelectronic devices to be stretchable and compliant for unobtrusive and high-fidelity signal acquisition. Thin conductive polymer film is regarded as an ideal interface; however, it is very challenging to simultaneously balance mechanical robustness and opto-electrical property. Here, a 40 nm-thick film based on photolithographic double-network conductive polymer mediated by graphene layer is reported, which concurrently enables stretchability, conductivity, and conformability. Photolithographic polymer and graphene endow the film photopatternability, enhance stress dissipation capability, as well as improve opto-electrical conductivity (4458 S cm-1@>90% transparency) through molecular rearrangement by π-π interaction, electrostatic interaction, and hydrogen bonding. The film is further applied onto corrugated facial skin, the subtle electromyogram is monitored, and machine learning algorithm is performed to understand complex emotions, indicating the outstanding ability for stretchable and compliant bioelectronics.

Rapid and Mechanically Robust Immobilization of Proteins on Silica Studied at the Single-Molecule Level by Force Spectroscopy and Verified at the Macroscopic Level.

Typical methods for stable immobilization of proteins often involve time-consuming surface modification of silicon-based materials to enable specific binding, while the nonspecific adsorption method is faster but usually unstable. Herein, we fused a silica-binding protein, Si-tag, to target proteins so that the target proteins could attach directly to silica substrates in a single step, markedly streamlining the immobilization process. The adhesion force between the Si-tag and glass substrates was determined to be approximately 400-600 pN at the single-molecule level by atomic force microscopy, which is greater than the unfolding force of most proteins. The adhesion force of the Si-tag exhibits a slight increase when pulled from the C-terminus compared to that from the N-terminus. Furthermore, the Si-tag's adhesion force on a glass surface is marginally higher than that on a silicon nitride probe. The binding properties of the Si-tag are not obviously affected by environmental factors, including pH, salt concentration, and temperature. In addition, the macroscopic adhesion force between the Si-tag-coated hydrogel and glass substrates was ∼40 times higher than that of unmodified hydrogels. Therefore, the Si-tag, with its strong silica substrate binding ability, provides a useful tool as an excellent fusion tag for the rapid and mechanically robust immobilization of proteins on silica and for the surface coating of silica-binding materials.

Identification of a novel CNV at the APC gene in a Chinese family with familial adenomatous polyposis.

Introduction: Familial adenomatous polyposis (FAP) is the second most commonly inherited colorectal cancer (CRC) predisposition caused by germline mutations within the adenomatous polyposis coli (APC) gene. The molecular defects and clinical manifestations of two FAP families were analyzed, and individual prevention strategies suitable for mutation carriers in different families were proposed. Methods and results: The pathogenic gene mutations were identified among the two families using whole-exome sequencing and verified with Sanger sequencing or quantitative polymerase chain reaction (qPCR). One novel (GRCh37:Chr5: 112145676-112174368, del, 28,692 bp) and a known (c.C847T:p.R283X) mutation in the APC gene were pathogenic mutations for FAP, according to the sequencing data and tumorigenesis pattern among the family members. The two mutations led to a premature translational stop signal, synthesizing an absent or disrupted protein product. Conclusion: Our findings expand the known germline mutation spectrum of the APC gene among the Chinese population. This reaffirms the importance of genetic testing in FAP. Genetic consultation and regular follow-ups are necessary for the individualized treatment of cancer-afflicted families with APC expression deficiency. Additional work is required to develop safe and effective chemotherapy and immunotherapy for FAP based on the mutation type.

Mechanically untying a protein slipknot: multiple pathways revealed by force spectroscopy and steered molecular dynamics simulations.

Protein structure is highly diverse when considering a wide range of protein types, helping to give rise to the multitude of functions that proteins perform. In particular, certain proteins are known to adopt a knotted or slipknotted fold. How such proteins undergo mechanical unfolding was investigated utilizing a combination of single molecule atomic force microscopy (AFM), protein engineering, and steered molecular dynamics (SMD) simulations to show the mechanical unfolding mechanism of the slipknotted protein AFV3-109. Our results reveal that the mechanical unfolding of AFV3-109 can proceed via multiple parallel unfolding pathways that all cause the protein slipknot to untie and the polypeptide chain to completely extend. These distinct unfolding pathways proceed via either a two- or three-state unfolding process involving the formation of a well-defined, stable intermediate state. SMD simulations predict the same contour length increments for different unfolding pathways as single molecule AFM results, thus providing a plausible molecular mechanism for the mechanical unfolding of AFV3-109. These SMD simulations also reveal that two-state unfolding is initiated from both the N- and C-termini, while three-state unfolding is initiated only from the C-terminus. In both pathways, the protein slipknot was untied during unfolding, and no tightened slipknot conformation was observed. Detailed analysis revealed that interactions between key structural elements lock the knotting loop in place, preventing it from shrinking and the formation of a tightened slipknot conformation. Our results demonstrate the bifurcation of the mechanical unfolding pathway of AFV3-109 and point to the generality of a kinetic partitioning mechanism for protein folding/unfolding.

Self-assembling branched amphiphilic peptides for targeted delivery of small molecule anticancer drugs.

Water insolubility poses a significant challenge in the clinical applications of many small molecule drugs. To improve the drug delivery efficiency, two branched amphiphilic peptides (BAPs) were designed in a computer-aided manner, for drug-loading through peptide self-assembling. The structures of the two BAPs, bis(LVFFA)-K-RGD (PepV-1) and bis(FHF)-K-RGD (PepV-2), were inspired by phospholipids, containing the RGD sequence as the hydrophilic head and two hydrophobic sequences as the hydrophobic tails. PepV-1 could self-assemble into nano-fibrils with a hydrophobic core and the RGD moiety on the surface. Its drug-loading efficiency (DE%) of three small molecule anticancer drugs (doxorubicin, camptothecin and curcumin) ranged from 9.90% to 11.74%, and entrapment efficiency (EE%) ranged from 37.30% to 43.00%. Pep-V2 could self-assemble into bilayer delimited nano-vesicles. The DE% of PepV-2 for these drugs ranged from 15.87% to 18.55%, and the EE% ranged from 60.45% to 73.23%. Both BAP carriers could prolong the release of the small molecule drugs, and the PepV-2 vesicles also showed pH-triggered increase of drug release due to the histidine residues. Bothe BAP carriers could increase the cytotoxicity against cancer cells, which might be due to the targeting on the cancer overexpressed integrins. The designed BAP carriers represent promising functional drug carriers for targeted drug delivery, and will be useful for improving the clinical use of small molecule drugs, especially for those with poor water solubility.

Pathogen-host adhesion between SARS-CoV-2 spike proteins from different variants and human ACE2 studied at single-molecule and single-cell levels.

The binding of the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein onto human angiotensin-converting enzyme 2 (ACE2) is considered as the first step for the virus to adhere onto the host cells during the infection. Here, we investigated the adhesion of spike proteins from different variants and ACE2 using single-molecule and single-cell force spectroscopy. We found that the unbinding force and binding probability of the spike protein from Delta variant to the ACE2 were the highest among the variants tested in our study at both single-molecule and single-cell levels. As the most popular variants, the Omicron variants have slightly higher unbinding force to the ACE2 than wild type. Molecular dynamics simulation showed that ACE2-RBD (Omicron BA.1) complex is destabilized by the E484A and Y505H mutations and stabilized by S477N and N501Y mutations, when compared with Delta variant. In addition, a neutralizing antibody, produced by immunization with wild type spike protein, could effectively inhibit the binding of spike proteins from wild type, Delta and Omicron variants (BA.1 and BA.5) onto ACE2. Our results provide new insight for the molecular mechanism of the adhesive interactions between spike protein and ACE2 and suggest that effective monoclonal antibody can be prepared using wild type spike protein against different variants.