LCB1 polypeptide mutant with enhanced affinity for SARS-CoV-2 Omicron pedigree variant, nucleic acid, composition and application
By constructing an LCB1 mutant library and using yeast surface display technology to screen for high-affinity LCB1 peptide mutants, the problem of decreased binding activity of LCB1 to Omicron mutants was solved, achieving efficient binding and broad-spectrum antiviral effects against multiple Omicron mutants.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-23
- Publication Date
- 2026-04-10
AI Technical Summary
The existing LCB1 peptides have reduced binding activity and neutralizing efficacy against SARS-CoV-2 Omicron lineage variants, and there is an urgent need to modify them to enhance their inhibitory ability against popular variants such as Omicron.
LCB1 mutant libraries were constructed using error-prone PCR technology. High-affinity LCB1 peptide mutants were screened using yeast surface display technology. Flow cytometry was then used for fine screening to obtain LCB1 peptide mutants with higher binding capacity to SARS-CoV-2 Omicron variants BA.1, BA.2, EG.5, and JN.1.
The LCB1 peptide significantly enhanced the affinity of the Omicron mutant strain, providing a broader antiviral spectrum. The process is feasible and has good development prospects.
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Figure CN121824701A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of protein engineering and antiviral technology, and particularly relates to an LCB1 polypeptide mutant with enhanced affinity for SARS-CoV-2 Omicron lineage variants, nucleic acids, compositions and applications. BACKGROUND
[0002] The novel coronavirus (SARS-CoV-2) and its continuously emerging variants, especially the Omicron lineage variants, pose a long-term threat to global public health. The interaction between the receptor binding domain (RBD) of the viral spike protein (Spike protein) and the angiotensin-converting enzyme 2 (ACE2) receptor of the host cell is a key link in viral infection, and thus becomes an important target for the development of antiviral drugs.
[0003] LCB1 is a microprotein composed of 56 amino acids, containing three different alpha helices. It simulates the binding of the RBD sequence with high affinity, thereby effectively blocking the interaction between RBD and ACE2. However, with the mutation of the virus, the Omicron variant has accumulated multiple mutations in its RBD region, resulting in varying degrees of decline in the binding activity and neutralizing potency of LCB1 to the variant. Therefore, it is urgent to modify LCB1 to enhance its inhibitory capacity for the Omicron and other prevalent variants.
[0004] Yeast surface display technology is a highly efficient protein engineering platform that can display the target protein as a fusion protein on the surface of yeast cells, and realize high-throughput quantitative screening and analysis by flow cytometry. Incubation of yeast cells with labeled targets can determine the binding of the target protein to the target. Standardizing the fluorescence intensity of the binding signal to the mean fluorescence intensity, and fitting with the target concentration as the variable, can determine the equilibrium dissociation constant (Kd) of the target protein. Studies have shown that the Kd values determined using yeast surface display are consistent with those measured using fluorescence polarization, surface plasmon resonance and biolayer interferometry. SUMMARY
[0005] The present application aims to overcome the deficiencies in the prior art and provide an LCB1 polypeptide mutant with enhanced affinity for SARS-CoV-2 Omicron lineage variants, nucleic acids, compositions and applications.
[0006] The technical solution adopted by the present application to solve its technical problems is as follows: An LCB1 polypeptide mutant with enhanced affinity for SARS-CoV-2 Omicron lineage variants, the amino acid sequence of the LCB1 polypeptide mutant being shown in SEQ ID NO. 2.
[0007] Further, the LCB1 polypeptide mutant has an amino acid sequence with at least 80% sequence identity to SEQ ID NO. 2, and is capable of binding to the receptor binding domain RBD of the SARS-CoV-2 Omicron variant spike protein.
[0008] Further, the LCB1 polypeptide mutant has higher affinity to the sub-variants BA.1, BA.2, EG.5, JN.1 of SARS-CoV-2 Omicron.
[0009] The method for obtaining the LCB1 polypeptide mutant as described above comprises the following steps: (1) Mutant library construction and primary screening: using the gene encoding the wild-type LCB1 polypeptide as a template, random mutations are introduced by error-prone PCR technology to construct an in vitro mutant library; the library is displayed on the surface of yeast cells, and four rounds of FACS cell sorting are performed using the RBD of the SARS-CoV-2 Omicron BA.1, BA.2, EG.5, JN.1 variant with His tag in sequence, and positive clones capable of simultaneously binding to the four Omicron variants are screened out, and finally 45 preliminary mutants with potential broad-spectrum binding ability are obtained; (2) Fine screening and function verification: the binding strength of the 45 mutants to the RBD of the Omicron BA.1, BA.2, EG.5, JN.1 variant is quantitatively evaluated by flow cytometry, and a preferred mutant showing the strongest binding signal to the four Omicron variants is screened out by yeast surface titration.
[0010] A nucleic acid molecule encoding the LCB1 polypeptide mutant as described above.
[0011] A pharmaceutical composition comprising a therapeutically effective amount of the LCB1 polypeptide mutant as described above.
[0012] A pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid molecule as described above.
[0013] The LCB1 polypeptide mutant as described above for use in the preparation of a medicament for preventing and treating SARS-CoV-2 infection.
[0014] The nucleic acid molecule as described above for use in the preparation of a medicament for preventing and treating SARS-CoV-2 infection.
[0015] The pharmaceutical composition as described above for use in the preparation of a medicament for preventing and treating SARS-CoV-2 infection.
[0016] The advantages and positive effects obtained by the present application are: 1. Affinity enhancement: The present application obtains mutants with significantly enhanced affinity through the strategy of combining directed evolution with rational design, providing a solid foundation for their in vitro and in vivo neutralization potency.
[0017] 2. Broad-spectrum potential: The mutant polypeptides provided by the present application exhibit stronger binding capacity to BA.1, BA.2, EG.5, JN.1 and other Omicron variants than wild-type LCB1, and have a broader antiviral spectrum.
[0018] 3. Feasible process: The polypeptides of the present application can be produced in large quantities through genetic engineering, and the process is relatively mature, with good development prospects.
[0019] 4. The polypeptide of the present application is obtained by the following method: error-prone PCR is used to construct an LCB1 mutation library, yeast surface display technology is used, and RBD proteins of SAS-CoV-2 Omicron BA.1, BA.2, EG.5, JN.1 variants are used for four rounds of FACS cell sorting to obtain preliminary mutants. Then, flow cytometry is used to screen mutant polypeptides that have good binding signals with RBD of SARS-COV-2 Omicron BA.1, BA.2, EG5, JN.1 variants. The amino acid sequence of the polypeptide is shown in LCB1 M (i.e. SEQ ID NO. 2). Through yeast surface titration experiment determination, the affinity of the mutant to various SARS-CoV-2 Omicron variant RBDs is significantly better than that of wild-type LCB1. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 is the result of E. coli electroporation in the present application; wherein, Figure A is the dilution of 1000 times of the electroporation bacterial liquid for plate coating; Figure B is the dilution of 100 times of the electroporation bacterial liquid for plate coating; Figure 2 is the result of yeast EBY100 transformation in the present application; wherein, Figure A is the dilution of 1000 times of the transformation bacterial liquid for plate coating; Figure B is the dilution of 100 times of the transformation bacterial liquid for plate coating; Figure 3 is the result of yeast surface titration in the present application; wherein, Figure A is the Kd value of wild-type LCB1 and five kinds of RBD; Figure B is the Kd value of LCB1 M and five kinds of RBD. DETAILED DESCRIPTION
[0021] The present application will be further described in conjunction with the examples below, which are descriptive and not limiting, and cannot limit the protection scope of the present application by the following examples.
[0022] The various experimental operations involved in the embodiments are conventional techniques in the art, and the parts not specifically annotated herein can be implemented by referring to various commonly used tool books, scientific and technical literature or related instructions, manuals, etc. before the application date of the present application.
[0023] A LCB1 polypeptide mutant with enhanced affinity to SARS-CoV-2 Omicron lineage variants, the amino acid sequence of which is shown in SEQ ID NO. 2.
[0024] Further, the LCB1 polypeptide mutant has an amino acid sequence with at least 80% sequence identity to SEQ ID NO. 2, and is capable of binding to the receptor binding domain RBD of the SARS-CoV-2 Omicron variant spike protein.
[0025] Further, the LCB1 polypeptide mutant has higher affinity to the subvariants BA.1, BA.2, EG.5, JN.1 of SARS-CoV-2 Omicron.
[0026] A method for obtaining the LCB1 polypeptide mutant as described above, comprising the following steps: (1) Mutant library construction and primary screening: using the gene encoding the wild-type LCB1 polypeptide as a template, random mutations are introduced by error-prone PCR technology to construct an in vitro mutant library; the library is displayed on the surface of yeast cells, and four rounds of FACS cell sorting are performed using RBD proteins of SARS-CoV-2 Omicron BA.1, BA.2, JN.1, and EG.5 variants with His tags, respectively, to screen out positive clones capable of simultaneously binding to the four Omicron variants, and finally 45 preliminary mutants with potential broad-spectrum binding ability are obtained; (2) Fine screening and function verification: the binding strength of the 45 mutants to BA.1, BA.2, EG.5, and JN.1 variant RBD proteins is quantitatively evaluated using a flow cytometer, and a preferred mutant that exhibits the strongest binding signal to all four Omicron variants is screened out using yeast surface titration.
[0027] A nucleic acid molecule encoding the LCB1 polypeptide mutant as described above.
[0028] A pharmaceutical composition comprising a therapeutically effective amount of the LCB1 polypeptide mutant as described above.
[0029] A pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid molecule as described above.
[0030] Use of the LCB1 polypeptide mutant as described above in the preparation of a medicament for the prevention and treatment of SARS-CoV-2 infection.
[0031] Use of the nucleic acid molecule as described above in the preparation of a medicament for the prevention and treatment of SARS-CoV-2 infection.
[0032] Use of the pharmaceutical composition as described above in the preparation of a medicament for the prevention and treatment of SARS-CoV-2 infection.
[0033] Specifically, the preparation and detection are as follows: LCB1 amino acid sequence (SEQ ID NO. 1): DKEWILQKIYEIMRLLDELGHAEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER LCB1 M Amino acid sequence (SEQ ID NO. 2): AKEWILQKIYEIMILLDELGHGEASMRVSDLIYEFMKKGDERLLEEAERLLEEVER.
[0034] Example 1: Construction of LCB1 mutant library and yeast surface display Using the polypeptide LCB1 encoding gene as a template, a degenerate primer was designed, and random mutations were introduced by error-prone PCR to construct an LCB1 mutant gene library. The PCR product was ligated with the yeast surface display vector pCTCON2 by homologous recombination, and then electrotransformed into DH5a competent cells to prepare a high-diversity plasmid library. The transformation results are shown in Figure 1 Single colony growth was observed on ampicillin-resistant screening plates, indicating that the recombinant plasmid was successfully transferred into DH5a competent cells.
[0035] The above plasmid library was introduced into Saccharomyces cerevisiae EBY100 competent cells by chemical transformation method, spread on SD-CAA plates, and cultured in a 30°C constant temperature incubator for 2-3 days. The transformation results are shown in Figure 2 Clear single colonies were formed on the SD-CAA selective medium, indicating that the recombinant plasmid was successfully transferred into yeast EBY100. Single colonies were picked and cultured in SD-CAA liquid medium at 30°C in a constant temperature shaker overnight for amplification. Then SG-CAA liquid medium was used, and the yeast was induced at 20°C for 48 h to display LCB1 mutants on the yeast surface.
[0036] Example 2: FACS-based multi-variant RBD binding clone screening The yeast cells successfully displaying LCB1 mutants were incubated with RBD of SARS-CoV-2 Omicron BA.1, BA.2, EG.5, JN.1 variants in a rotary mixer at 4°C overnight. The incubated cells were washed with PBS, centrifuged at 13000 rpm for 1 min, the supernatant was discarded, and the step was repeated once. The washed yeast cells were incubated with flow cytometry antibodies for detecting display level and detecting target protein binding at 4°C for 1 h in the dark, and then sorted using a flow cytometer. The strongest binding signal was collected by setting a gate to collect about 0.1-1% of the yeast cell population. The sorted cells were inoculated in SD-CAA liquid medium for recovery and expansion, and the above display and screening process was repeated to enrich yeast cells with continuously improved affinity.
[0037] Example 3: Identification and sequencing of candidate clones The yeast cells of the fourth round of screening were plated on SD-CAA plates, and single colonies were randomly picked for yeast colony PCR. The PCR products were sent to the company for sequencing. According to the sequencing results, the original sequence was compared to obtain the amino acid sequences of 45 mutants.
[0038] Example 4: Flow cytometry binding evaluation of candidate mutants The 45 mutants obtained in Example 3 were displayed on the surface of yeast EBY100, and the method was the same as in Example 1. They were incubated with RBD of SARS-CoV-2 Omicron BA.1, BA.2, EG.5, JN.1 variants in a rotary mixer at 4°C overnight. The incubated yeast cells were washed with PBS, centrifuged at 13000 rpm for 1 min at 4°C, and the step was repeated once. The washed cells were incubated with flow cytometry antibodies for detecting display level and detecting target protein binding at 4°C for 1 h in the dark, and then analyzed using a flow cytometer. The binding ability of each mutant was quantified and compared by calculating the ratio of the average fluorescence intensity (MFI) of the binding signal to the display signal. According to the results, 5 mutants with good binding to the four RBDs were selected. Further, the 5 mutants were incubated with different concentration gradients of RBD, labeled with flow cytometry antibodies, and the apparent affinity was measured by flow cytometry. Finally, the LCB1 mutant with the best binding performance was selected. M The yeast titration results of the mutant polypeptides with SARS-CoV-2 wild type and RBD of Omicron BA.1, BA.2, EG.5, JN.1 variants are shown in Figure 3
[0039] Figure 3 A is the Kd value of wild type LCB1 with SARS-CoV-2 WT and five mutant strains of Omicron BA.1, BA.2, EG.5, JN.1 RBD determined by yeast surface titration. Among them, the Kd value of LCB1 with SARS-CoV-2 wild type RBD is 0.359 nM. The Kd values of LCB1 with Omicron BA.1 RBD and BA.2 RBD are 1.168 nM and 2.104 nM, respectively. In addition, the affinity of LCB1 with Omicron EG.5 RBD and JN.1 RBD is weak, and there is almost no binding signal.
[0040] Figure 3 B is the Kd value of LCB1 M with SARS-CoV-2 WT and five mutant strains of Omicron BA.1, BA.2, EG.5, JN.1 RBD determined by yeast surface titration. The Kd values of LCB1 M with five RBDs are 0.171 nM, 0.125 nM, 0.351 nM, 41.77 nM and 166.1 nM, respectively. The mutant polypeptide shows significantly enhanced binding activity to each of the above mutant RBDs under the same conditions. Among them, the Kd values of LCB1 with WT, BA.1 and BA.2 mutant RBDs are increased by 2 times, 9 times and 6 times, respectively. In addition, compared with the case that LCB1 has no obvious binding with EG.5 and JN.1 mutant RBDs, LCB1 M maintains nanomolar level affinity for EG.5 and JN.1 mutant RBDs, showing superior broad-spectrum binding ability. The data show that the mutant polypeptide has the potential to be developed as a broad-spectrum anti-COVID-19 drug.
[0041] Although the embodiments of the present application are disclosed for illustrative purposes, those skilled in the art can understand that various alternatives, changes and modifications are possible without departing from the spirit and scope of the present application and the appended claims, therefore, the scope of the present application is not limited to the disclosed content of the embodiments.
Claims
1. An LCB1 polypeptide mutant with enhanced affinity for SARS-CoV-2 Omicron lineage variants, characterized in that: The amino acid sequence of the LCB1 polypeptide mutant is shown in SEQ IQ NO.
2.
2. The LCB1 polypeptide mutant according to claim 1, characterized in that: The LCB1 polypeptide mutant has an amino acid sequence that is at least 80% identical to that of SEQ IQ NO.2 and is capable of binding to the receptor-binding domain (RBD) of the spike protein of the SARS-CoV-2 Omicron variant.
3. The LCB1 polypeptide mutant according to claim 1, characterized in that: The LCB1 polypeptide mutant exhibits higher affinity for the subvariants BA.1, BA.2, EG.5, and JN.1 of SARS-CoV-2 Omicron.
4. The method for obtaining the LCB1 polypeptide mutant according to any one of claims 1 to 3, characterized in that: Includes the following steps: (1) Construction and initial screening of mutant library: Using the gene encoding wild-type LCB1 peptide as a template, random mutations were introduced by error-prone PCR technology to construct an in vitro mutant library; the library was displayed on the surface of yeast cells, and four rounds of cell sorting were performed using the RBD protein of SARS-CoV-2 Omicron BA.1, BA.2, EG.5 and JN.1 variants with His tag to screen positive clones that could bind to the four Omicron variants at the same time, and finally 45 preliminary mutants with potential broad-spectrum binding ability were obtained; (2) Fine screening and functional verification: The binding strength of these 45 mutants to the four RBD proteins BA.1, BA.2, EG.5 and JN.1 was quantitatively evaluated by flow cytometry. A preferred mutant that showed the strongest binding signal to all four Omicron mutant strains was selected by yeast surface titration.
5. A nucleic acid molecule encoding an LCB1 polypeptide mutant as described in any one of claims 1 to 3.
6. A pharmaceutical composition comprising a therapeutically effective amount of the LCB1 polypeptide mutant as claimed in any one of claims 1 to 3.
7. A pharmaceutical composition comprising a therapeutically effective amount of the nucleic acid molecule as claimed in claim 5.
8. The use of the LCB1 polypeptide mutant as described in any one of claims 1 to 3 in the preparation of a medicament for the prevention and treatment of SARS-CoV-2 infection.
9. The use of the nucleic acid molecule as described in claim 5 in the preparation of a medicament for the prevention and treatment of SARS-CoV-2 infection.
10. The use of the pharmaceutical composition of claim 6 or 7 in the preparation of a medicament for the prevention and treatment of SARS-CoV-2 infection.