SARS-CoV-2 affinity polypeptide inhibitor and application thereof

Abstract

The invention discloses an SARS-CoV-2 affinity polypeptide inhibitor and an application of the SARS-CoV-2 affinity polypeptide inhibitor. The invention provides a polypeptide capable of inhibiting SARS-CoV-2. The amino acid sequence of the polypeptide is as shown in SEQ ID No. 1; the invention also provides a polymer of the polypeptide and a derived fusion protein (which is formed by fusing the polypeptide / polypeptide polymer and an Fc fragment and / or tag protein and / or other functional proteins connected to an amino terminal or a carboxyl terminal of the polypeptide / polypeptide polymer). The invention provides a possible means for treating the SARS-CoV-2 infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an affibody polypeptide inhibitor of SARS-CoV-2 and applications thereof. Background Art

[0002] Coronavirus is a type of single-stranded positive-strand non-segmented RNA virus with an envelope. It is called coronavirus because the spikes on its envelope extend out in all directions in a crown shape. The coronavirus subfamily is divided into four genera based on their genotypes and hemotyping, namely α, β, γ and δ coronaviruses. Coronaviruses can infect a variety of hosts such as mammals and birds. Currently, the coronaviruses known to cause human infection are from the α-CoV and β-CoV genera, including HCoV-229E, HCoV-NL63, HCoV-oc43, HCoV-HKU1, SARS-CoV and MERS-CoV.

[0003] In December 2019, SARS-CoV-2 began to spread. Infection with the virus can cause symptoms such as fever, cough, shortness of breath, and difficulty breathing. In severe cases, it can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death.

[0004] Peptides are highly effective and low in toxicity, and have great potential as therapeutic drugs. Targeting the SARS-CoV-2S protein to screen for peptides that target pathogens that invade cells is the basis for the development of SARS-CoV-2 therapeutic drugs. Although there are currently a variety of vaccines and monoclonal antibodies against SARS-CoV-2, most studies have shown that their protective effect against the currently widely spread SARS-CoV-2 mutations has decreased, so a broader spectrum of new coronavirus drugs is urgently needed to cope with new epidemics that may occur in the future. Summary of the invention

[0005] The present invention claims protection for an affinity polypeptide inhibitor of SARS-CoV-2 and its application.

[0006] In a first aspect, the present invention claims a polypeptide capable of inhibiting SARS-CoV-2.

[0007] The polypeptide capable of inhibiting SARS-CoV-2 claimed in the present invention is an Affibody molecule, which consists of 58 amino acid residues in total, and its amino acid sequence is shown in SEQ ID No.1.

[0008] In a second aspect, the present invention claims protection for a polypeptide polymer capable of inhibiting SARS-CoV-2.

[0009] The polypeptide multimer capable of inhibiting SARS-CoV-2 claimed in the present invention is a multimer formed by connecting two or more polypeptides described in the first aspect above through a flexible peptide.

[0010] The flexible peptide is a soft, linear, and easily bendable amino acid sequence.

[0011] In a specific embodiment of the present invention, the flexible peptide is specifically GGGGSGGGGSGGG.

[0012] In a specific embodiment of the present invention, the polypeptide multimer is a dimer formed by connecting two polypeptides shown in SEQ ID No. 1 through a flexible peptide (GGGGSGGGGSGGG). Specifically, the amino acid sequence of the polypeptide multimer is shown in SEQ ID No. 3 (polypeptide dimer).

[0013] In a third aspect, the present invention claims protection for a fusion protein capable of inhibiting SARS-CoV-2.

[0014] The fusion protein capable of inhibiting SARS-CoV-2 claimed in the present invention may be any of the following:

[0015] (A1) is formed by fusing the polypeptide described in the first aspect above with an Fc fragment and / or a tag protein and / or other functional proteins connected to the amino terminus or carboxyl terminus of the polypeptide.

[0016] (A2) is formed by fusing the polypeptide multimer described in the second aspect above with an Fc fragment and / or a tag protein or other functional protein connected to the amino terminus or carboxyl terminus of the polypeptide multimer.

[0017] The tag protein is mainly used for purification, such as His tag, myc tag, Flag tag, HA tag, etc. The other functional proteins may be enzyme proteins with catalytic function or fluorescent proteins with fluorescent luminescence function.

[0018] Wherein, the Fc fragment may be derived from IgG, such as IgG1, IgG2, IgG3 or IgG4.

[0019] In a specific embodiment of the present invention, the Fc fragment is specifically an Fc fragment from human IgG1 (as shown in positions 59-291 of SEQ ID No. 2).

[0020] Specifically, the fusion protein in (A1) is formed by fusing the polypeptide shown in SEQ ID No.1 and the Fc fragment (positions 59-291 of SEQ ID No.2) connected to its carboxyl end. That is, the amino acid sequence of the fusion protein in (A1) is shown in SEQ ID No.2.

[0021] Specifically, the fusion protein in (A2) is formed by fusing the polypeptide multimer shown in SEQ ID No.3 and the Fc fragment (positions 59-291 of SEQ ID No.2) connected to its carboxyl end. That is, the amino acid sequence of the fusion protein in (A2) is shown in SEQ ID No.4.

[0022] In a fourth aspect, the present invention claims protection for a nucleic acid molecule encoding the polypeptide described in the first aspect above, the polypeptide multimer described in the second aspect above, or the fusion protein described in the third aspect above.

[0023] In a specific embodiment of the present invention, the nucleic acid molecule encoding the polypeptide (SEQ ID No.1) described in the first aspect above is shown as SEQ ID No.5.

[0024] In a specific embodiment of the present invention, the nucleic acid molecule encoding the polypeptide multimer (SEQ ID No.3) described in the second aspect above is shown as SEQ ID No.7.

[0025] In a specific embodiment of the present invention, the nucleic acid molecule encoding the fusion protein shown in SEQ ID No. 2 in the third aspect above is shown in SEQ ID No. 6.

[0026] In a specific embodiment of the present invention, the nucleic acid molecule encoding the fusion protein shown in SEQ ID No.4 in the third aspect above is shown in SEQ ID No.8.

[0027] In a fifth aspect, the present invention claims protection for an expression cassette or a recombinant vector or a recombinant microorganism or a transgenic cell line containing the nucleic acid molecule described in the fourth aspect above.

[0028] The expression cassette refers to a DNA capable of expressing the polypeptide described in the first aspect above, the polypeptide multimer described in the second aspect above, or the fusion protein described in the third aspect above in a host cell, and the DNA may include not only a promoter for initiating transcription of the target gene, but also a terminator for terminating transcription of the target gene. Further, the expression cassette may also include an enhancer sequence.

[0029] The vector may be a plasmid, cosmid, phage or viral vector.

[0030] The microorganism may be yeast, bacteria, algae, etc. The bacteria may be from Escherichia, Erwinia, Agrobacterium (such as Agrobacterium tumefaciens EHA105), Flavobacterium, Alcaligenes, Pseudomonas, Bacillus, etc.

[0031] In the sixth aspect, the present invention claims protection for a linker formed by covalent or non-covalent linkage between the polypeptide described in the first aspect above, the polypeptide polymer described in the second aspect above, or the fusion protein described in the third aspect above and a fluorescent group or a radioactive group or biotin or streptomycin or a nanomaterial.

[0032] In a seventh aspect, the present invention claims protection for any of the following applications:

[0033] (B1) Use of the polypeptide described in the first aspect above, or the polypeptide multimer described in the second aspect above, or the fusion protein described in the third aspect above, or the nucleic acid molecule described in the fourth aspect above, or the expression cassette or recombinant vector or recombinant microorganism or transgenic cell line described in the fifth aspect above, or the connector described in the sixth aspect above in the preparation of a product for inhibiting SARS-CoV-2;

[0034] (B2) Use of the polypeptide described in the first aspect above, or the polypeptide multimer described in the second aspect above, or the fusion protein described in the third aspect above, or the nucleic acid molecule described in the fourth aspect above, or the expression cassette or recombinant vector or recombinant microorganism or transgenic cell line described in the fifth aspect above, or the connector described in the sixth aspect above in the preparation of a product for treating and / or preventing diseases caused by SARS-CoV-2 infection;

[0035] (B3) Use of the polypeptide described in the first aspect above, or the polypeptide multimer described in the second aspect above, or the fusion protein described in the third aspect above, or the nucleic acid molecule described in the fourth aspect above, or the expression cassette or recombinant vector or recombinant microorganism or transgenic cell line described in the fifth aspect above, or the connector described in the sixth aspect above in the preparation of a product for improving symptoms caused by SARS-CoV-2 infection;

[0036] (B4) Use of the polypeptide described in the first aspect above, or the polypeptide multimer described in the second aspect above, or the fusion protein described in the third aspect above, or the nucleic acid molecule described in the fourth aspect above, or the expression cassette or recombinant vector or recombinant microorganism or transgenic cell line described in the fifth aspect above, or the connector described in the sixth aspect above in the preparation of a product capable of binding to the S protein of SARS-CoV-2;

[0037] (B5) Use of the polypeptide described in the first aspect above, or the polypeptide multimer described in the second aspect above, or the fusion protein described in the third aspect above, or the nucleic acid molecule described in the fourth aspect above, or the expression cassette or recombinant vector or recombinant microorganism or transgenic cell line described in the fifth aspect above, or the connector described in the sixth aspect above in the preparation of a product for diagnosing a disease caused by SARS-CoV-2 infection;

[0038] Furthermore, the SARS-CoV-2 is a prototype strain (SARS-CoV-2-PT) or a variant strain of SARS-CoV-2.

[0039] Furthermore, the SARS-CoV-2 variant strain may be Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron (BA.1), Omicron (BA.2), Omicron (BA.2.12.1), Omicron (BA.2.75), Omicron (BA.4 / 5), Omicron (BF.7), Omicron (BA.4.6), Omicron (BQ.1), Omicron (BQ.1.1), Omicron (XBB), Omicron (XBB.1.5) or Omicron (CH.1.1).

[0040] Beneficial effects of the invention: The present invention provides a polypeptide and polypeptide derivatives that can bind to the SARS-CoV-2 spike protein and neutralize the new coronavirus pseudovirus, which can be used as potential drugs for preparing diagnosis, prevention and treatment of diseases caused by SARS-CoV-2, with huge market value and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 These are the molecular sieve chromatography results and SDS-PAGE detection results of SARS-CoV-2S-6P protein.

[0042] Figure 2 The molecular sieve chromatography and SDS-PAGE detection results of Affibody polypeptide S-2 protein.

[0043] Figure 3 The molecular sieve chromatography results and SDS-PAGE detection results of Affibody peptide S-2-dimer protein.

[0044] Figure 4The molecular sieve chromatography and SDS-PAGE detection results of Affibody polypeptide S-2-Fc-1 protein.

[0045] Figure 5 The molecular sieve chromatography and SDS-PAGE detection results of Affibody polypeptide S-2-Fc-2 protein.

[0046] Figure 6 The kinetic curve results of Affibody peptide S-2 binding to SARS-CoV-2S-6P.

[0047] Figure 7 The neutralizing activity of Affibody peptides S-2, S2-Fc1, S2-dimer, S2-Fc2 and SARS-CoV-2 prototype strain pseudovirus.

[0048] Figure 8 The neutralizing activity of Affibody peptide S2-Fc2 and SARS-CoV-2 prototype and mutant pseudoviruses. DETAILED DESCRIPTION

[0049] The present invention uses mRNA display technology to screen Affibody polypeptide drugs for preventing or treating SARS-CoV-2.

[0050] SARS-CoV-2 spike (S) protein plays an important role in mediating the recognition and membrane fusion of viruses and cells. S protein is divided into S1 subunit and S2 subunit according to different functions; wherein the S1 subunit is located at the amino terminus of the S protein, which is the key site for recognition with the cell membrane ACE2 receptor and the main target of neutralizing antibodies. The S2 subunit can fix the S protein on the cell membrane and mediate the fusion of the viral envelope with the host cell membrane. The present invention screens Affibody polypeptides for the extracellular domain protein of the S protein. An Affibody-mRNA library is constructed, and a specific polypeptide drug inhibitor S-2 of SARS-CoV-2 and a derivative polypeptide of S-2 (S-2-dimer, S-2-Fc-1, S-2-Fc-2) are screened out by mRNA in vitro display technology.

[0051] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0052] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0053] The experimental instruments and materials used in the following examples are:

[0054] HEK293F cells (from ATCC cell bank).

[0055] HEK293T cells (from ATCC cell bank).

[0056] Vero-E6 cells (from ATCC cell bank).

[0057] The pseudovirus was packaged with backbone virus VSV-ΔG-EGFP (product of Wuhan Shumi Brain Science and Technology Co., Ltd., catalog number V03001).

[0058] Eukaryotic system protein pCAGGS and pET-21a expression vectors (synthesized by Suzhou GeneWeizhi Company).

[0059] Example 1. Expression and purification of SARS-CoV-2S-6P protein

[0060] In 2020, Jason S. McLellan published an article in Science: Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. In this article, in order to solve the problem that the prefusion conformation of the new crown S protein is extremely unstable and difficult to recombinantly express in mammalian cells, they continued to make structural modifications on the basis of the original S-2P, designed 100 S protein candidate sequences, optimized the combination, and finally designed HexaPro with 6 proline mutations, which significantly improved the production and stability of the S protein, and the electron microscopy structure confirmed that the 6 proline mutations can lock the S protein in the prefusion conformation. The S protein mentioned in the specification of the present invention is the S protein after 6 proline mutations, and the S protein after mutation is more stable.

[0061] First, the S gene was fused with the His tag (its amino acid sequence was from NCBI Reference Sequence: YP_009724390.1), the sequence was codon-optimized and then artificially synthesized, and then linked to the pCAGGS expression vector (the vector was synthesized by Suzhou Jinweizhi Company). The resulting recombinant vector was named pCAGGS-S-6P. Structural description of the recombinant vector pCAGGS-S-6P: The recombinant plasmid obtained by inserting the DNA fragment shown in SEQ ID No.9 between EcoRⅠ and XholⅠ of the pCAGGS vector.

[0062] Protein expression and purification were performed as follows:

[0063] a) Cell preparation: Mammalian HEK293F cells were passaged in cell flasks one day in advance and incubated at 37°C and 5% CO 2 Culture in an incubator for 12-24 hours, so that the cell density reaches about 2.0×10 6 / mL.

[0064] b) Transfection: The co-transfection density was 2×10 6 / mL HEK293F cells. Dilute the plasmid with 150mM sodium chloride solution (1mL cell solution plus 1μg pCAGGS-S-6P plasmid), dilute 1mg / mL PEI with 150mM sodium chloride solution (1mL cell solution plus 3μg PEI), and let the solution stand for 5min; mix the above two and let stand for 15min, then add dropwise to HEK293F cells. Add feed solution (Sino Biological, M293-SUPI) at a ratio of 1L to 35mL at 24h and 72h after transfection, and culture in a 37℃ incubator for 96h.

[0065] c) Protein purification: Use a 4°C high-speed centrifuge, centrifuge at 8000 rpm for 1 hour to collect the cell supernatant, filter through a 0.22 μm sterile filter membrane, and bind to a His pre-packed column (Situofan, 17-5248-01). Wash away impurities with 20 mM Tirs, 200 mM NaCl, 20 mM imidazole (pH 8.0), and elute the bound target protein with 20 mM Tirs, 200 mM NaCl, 300 mM imidazole (pH 8.0). Collect and concentrate the protein and pass it through a Superose TM 6Increase10 / 300GL (Situofan, 29091596) was purified by gel filtration chromatography. The target protein was confirmed by gel filtration chromatography peak position and SDS-PAGE. The results are as follows Figure 1 As shown, the purified S-6P protein has good homogeneity and high purity.

[0066] Example 2: Expression and purification of Affibody polypeptides

[0067] In order to obtain an Affibody polypeptide inhibitor specific for human SARS-CoV-2S protein, screening was performed by mRNA display technology. Finally, the Affibody polypeptide S-2 was obtained, and its amino acid sequence is shown in SEQ ID No.1. And based on its design, the polypeptide S-2-dimer was derived, which is a dimer form of the Affibody polypeptide S-2, and two polypeptides S-2 are connected by GS-linker. The amino acid sequence of S-2-dimer is shown in SEQ ID No.3. Furthermore, S-2 and S-2-dimer fusion Fc fragments (from human IgG1) were designed respectively, and the S-2 fusion Fc fragment was named S-2-Fc-1, and the S-2-dimer fusion Fc fragment was named S-2-Fc-2. The amino acid sequence of S-2-Fc-1 is shown in SEQ ID No.2, and the amino acid sequence of S-2-Fc-2 is shown in SEQ ID No.4.

[0068] The screened Affibody polypeptide S-2 (SEQ ID No. 1) and its derivative polypeptide S-2-dimer (SEQ ID No. 3) were expressed and purified using a prokaryotic expression system, and the screened Affibody polypeptide S-2 derivative polypeptides S-2-Fc-1 (SEQ ID No. 2) and S-2-Fc-2 (SEQ ID No. 4) were expressed and purified using a mammalian cell expression system.

[0069] The specific operations are as follows:

[0070] 1. Construction of recombinant expression vector

[0071] The Affibody polypeptide S-2 and S-2-dimer genes were fused with the His tag for expression, and the sequences were artificially synthesized after codon optimization to obtain the optimized S-2 gene (SEQ ID No.5) and the optimized S-2-dimer gene (SEQ ID No.7). Then, the optimized S-2 gene and the optimized S-2-dimer gene were connected to the pET-21a expression vector (the vector was synthesized by Suzhou Jinweizhi Company). After sequencing verification, the recombinant expression vectors pET-21a-S-2 and pET-21a-S-2-dimer were finally obtained.

[0072] Structural description of the recombinant expression vector pET-21a-S-2: The recombinant plasmid obtained by inserting the optimized S-2 gene shown in SEQ ID No.5 between the restriction sites NdeⅠ and XholⅠ of pET-21a.

[0073] Structural description of the recombinant expression vector pET-21a-S-2-dimer: The recombinant plasmid obtained by inserting the optimized S-2-dimer gene shown in SEQ ID No.7 between the restriction sites NdeⅠ and XholⅠ of pET-21a.

[0074] The Affibody polypeptide S-2 derived polypeptide S-2-Fc-1 and S-2-Fc-2 gene sequences were artificially synthesized after codon optimization to obtain optimized S-2-Fc-1 (SEQ ID No.6) and optimized S-2-Fc-2 genes (SEQ ID No.8). Then, the optimized S-2-Fc-1 gene and optimized S-2-Fc-2 gene were connected to the pCAGGS expression vector (the vector was synthesized by Suzhou Jinweizhi Company). After sequencing verification, the recombinant expression vectors pCAGGS-S-2-Fc-1 and pCAGGS-S-2-Fc-2 were finally obtained.

[0075] Structural description of the recombinant expression vector pCAGGS-S-2-Fc-1: The optimized S-2-Fc-1 gene shown in SEQ ID No. 6 is inserted between the restriction sites EcoRⅠ and XholⅠ of pCAGGS to obtain a recombinant plasmid.

[0076] Structural description of the recombinant expression vector pCAGGS-S-2-Fc-2: The optimized S-2-Fc-2 gene shown in SEQ ID No. 8 is inserted between the restriction sites EcoRⅠ and XholⅠ of pCAGGS to obtain a recombinant plasmid.

[0077] 2. Protein expression and purification in prokaryotic system

[0078] (1) Transformation: The plasmids of the Affibody peptide S-2 and the derivative peptide S-2-dimer of S-2 synthesized in the previous step (pET-21a-S-2 and pET-21a-S-2-dimer) were diluted to 100 ng / μL with sterile water, and 1 μL of the plasmid was transformed into BL21 (DE3) competent cells by heat shock method, followed by amplification with 500 μL of sterile LB medium, and then an appropriate amount of bacterial solution was spread on a solid plate containing ampicillin resistance, and cultured in a 37°C incubator for about 24 h, and obvious white monoclonal colonies appeared.

[0079] (2) Activation and recovery: Pick a single clone from the transformed plate and culture it in 5 mL of LB medium containing ampicillin resistance at 37°C in a constant temperature shaker for about 12 hours to fully activate and recover the bacteria. Generally, two single clones are picked and performed simultaneously to ensure the positive rate and repeatability.

[0080] (3) Bacterial liquid expansion: Transfer the activated bacterial liquid to 50 mL of new culture medium at a ratio of 1:50 and place it in a 37°C shaker for culture. After 2 hours, transfer the bacterial liquid to 1000 mL of new culture medium at a ratio of 1:50 and place it in a 37°C shaker for culture for about 2.5 hours. During this period, pay attention to the growth of bacteria and use a spectrophotometer to monitor the absorbance value of the bacterial liquid at a wavelength of 600 nm (OD600). When it is controlled between 0.4-0.8, it can be considered that the bacterial liquid is in the logarithmic growth phase.

[0081] (4) Induction: The bacterial solution in the logarithmic growth phase was induced with a final concentration of 1 mM IPTG and cultured in a 37°C constant temperature incubator for 5 h.

[0082] (5) Protein purification: Use a 4°C high-speed centrifuge and centrifuge at 1,2000 rpm for 10 minutes to collect the bacteria and discard the culture medium supernatant. Then resuspend the bacteria in 50 mL of 1×PBS and then lyse the bacteria with an ultrasonic disruptor. The parameters are usually set as follows: 200 watts, ultrasonic for 5 seconds, stop for 9 seconds, and a total time of 30 minutes. Use a 4°C high-speed centrifuge to centrifuge the completely disrupted bacterial solution at 1,2000 rpm for 30 minutes to collect the supernatant, filter through a 0.22 μm sterile filter membrane, and purify by His affinity chromatography and Superdex 75 10 / 300GL gel filtration chromatography. The peak position of the target protein was determined by SDS-PAGE, and the results are as follows. Figure 2 , Figure 3 As shown, the purified protein has high purity and good homogeneity.

[0083] 3. Mammalian system protein expression and purification

[0084] (1) Cell preparation: Mammalian HEK293F cells were passaged one day in advance and incubated at 37°C and 5% CO 2 Culture in an incubator for 12-24 hours, so that the cell density reaches about 2.0×10 6 / mL.

[0085] (2) Transfection: The target plasmid (the recombinant expression vectors pCAGGS-S-2-Fc-1 and pCAGGS-S-2-Fc-2 constructed in step 1) and PEI were mixed at a ratio of 1:3 and then transiently transfected into HEK293F cells to express the Affibody peptide S-2 derivative peptides S-2-Fc-1 and S-2-Fc-2 proteins. Feed solution (Sino Biological, M293-SUPI) was added 24 h and 72 h after transfection, and the cells were cultured in a 37°C incubator for 96 h.

[0086] (3) Protein purification: Use a 4°C high-speed centrifuge and centrifuge at 8000 rpm for 1 h to collect the cell supernatant, filter it through a 0.22 μm sterile filter membrane, and purify it by HiTrap Protein A HP (Situofan, 17040501) affinity chromatography and Superdex 200 10 / 300GL (Situofan, GE28-9909-44) gel filtration chromatography. The target protein was determined by gel filtration chromatography peak position and SDS-PAGE. The results are as follows: Figure 4 , Figure 5 As shown, the purified protein has high purity and good homogeneity.

[0087] Example 3. Affinity determination of Affibody peptide S-2 and SARS-CoV-2 S-6P protein

[0088] The new crown S-6P protein (prepared in Example 1) was diluted to 50 μg / mL with 0.01M sodium acetate solution (pH 4.5) and fixed on a CM5 chip (Cytiva, Sensor Chip CM5) by amino coupling. The Affibody polypeptide S-2 (prepared in Example 2) was diluted stepwise with PBST solution (PBS buffer containing 0.05‰ Tween 20, volume percentage) to different concentrations (1600, 800, 400, 200, 100nM) of the test solution, each concentration volume is 100μL. Using Biacore 8K (GE) using the Single-cycle kinetics method, the sample position is displayed according to the preset program, the sample is added to a 96-well plate (GE), and the binding of different concentrations of Affibody polypeptide S-2 and the new crown S-6P protein is detected, and the chip is regenerated using 5mM NaOH.

[0089] The results are as follows Figure 6 As shown, it can be seen that Affibody peptide S-2 can bind to the new coronavirus S-6P protein with an affinity of 43.9nM.

[0090] Example 4: Packaging of SARS-CoV-2 Prototype and Variant Pseudoviruses

[0091] The present invention prepares the following 17 pseudoviruses: pseudoviruses of the SARS-CoV-2 prototype strain (SARS-CoV-2-PT) and 16 variant strains (Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron (BA.1), Omicron (BA.2), Omicron (BA.2.12.1), Omicron (BA.2.75), Omicron (BA.4 / 5), Omicron (BF.7), Omicron (BA.4.6), Omicron (BQ.1), Omicron (BQ.1.1), Omicron (XBB), Omicron (XBB.1.5) and Omicron (CH.1.1).

[0092] Among them, the pseudoviruses of the SARS-CoV-2 prototype strain (SARS-CoV-2-PT) and variant strains B.1.1.7, B.1.351, P.1, and B.1.617.2 are recorded in the article “Zhao X, Zheng A, Li D, Zhang R, Sun H, Wang Q, Gao GF, Han P, Dai L. Neutralisation of ZF2001-elicited antisera to SARS-CoV-2 variants. Lancet Microbe. 2021Oct;2(10):e494.doi:10.1016 / S2666-5247(21)00217-2.Epub 2021Aug 20.PMID:34458880;PMCID:PMC8378832”, in which the SARS-CoV-2 prototype strain is referred to as “SARS-CoV-2wild” in the article. type" is available to the public from the applicant and may only be used to repeat the experiments of the present invention and may not be used for any other purpose.

[0093] The pseudovirus of SARS-CoV-2 mutant strain BA.1 is recorded in "Huang M, Wu L, Zheng A, Xie Y, He Q, Rong X, Han P, Du P, Han P, Zhang Z, Zhao R, Jia Y, Li L, Bai B, Hu Z, Hu S, Niu S, Hu Y, Liu H, Liu B, Cui K, Li W, Zhao GF.Atlas of currently available human neutralizing antibodies against SARS-CoV-2and escape byOmicron sub-variantsBA.1 / BA.1.1 / BA.2 / BA.3.Immunity.2022Aug 9;55(8):1501-1514.e3.doi:10.1016 / j.immuni.2022.06.005.Epub 2022Jun 15.PMID:35777362;PMCID:PMC9197780" is available to the public from the applicant and can be used to repeat the experiments of the present invention and cannot be used for other purposes.

[0094] The pseudoviruses of SARS-CoV-2 mutant strains BA.2, BA.2.12.1, BA.2.75, BA.4 / 5, BQ.1.1, and XBB are recorded in "He Q, Wu L, Xu Z, Wang X, Xie Y, Chai Y, Zheng A, Zhou J, Qiao S, Huang M, ShangG, Zhao atlas of antibody evasion by SARS-CoV-2Omicron sub-variants including BQ.1.1and 21.PMID:37019110;PMCID:PMC10027947" is available to the public from the applicant and can be used to repeat the experiments of the present invention and cannot be used for other purposes.

[0095] The remaining pseudoviruses of several SARS-CoV-2 variants were prepared as follows:

[0096] Due to the truncation of the last 18 amino acids of the S protein C-terminus, it was found that the virus packaging and infection efficiency of VSV-SARS-CoV-2-Sdel18 (S protein with 18 amino acids truncated at the carboxyl terminus) was much higher than that of VSV-SARS-CoV-2-S. https: / / www.antpedia.com / ibook42 / n / 82911-n.html (reference: Xiong HL, Wu YT, Cao JL, et al. Robust neutralization assay based on SARS-CoV-2S-protein-bearing vesicularstomatitis virus (VSV) pseudovirus and ACE2-overexpressing BHK21 cells. Emerg Microbes Infect. 2020). Therefore, the truncated S protein expression plasmid was used in this embodiment to package the SARS-CoV-2 prototype strain and variant strain pseudovirus.

[0097] 1. Preparation of expression plasmid for truncated S protein

[0098] (1) After removing the nucleotides encoding the last 18 amino acids of the S protein of the mutant strains Omicron (BF.7), Omicron (BA.4.6), Omicron (BQ.1), Omicron (XBB.1.5), and Omicron (CH.1.1), the nucleotide sequences BF.7-S-del18 (SEQ ID No.10), BA.4.6-S-del18 (SEQ ID No.11), BQ.1-S-del18 (SEQ IDNo.12), XBB.1.5-S-del18 (SEQ IDNo.13), and CH.1.1-S-del18 (SEQ IDNo.14) were obtained, respectively, and synthesized by GENEWIZ.

[0099] (2) The nucleotide sequences obtained in (1) were cloned into the restriction sites EcoRⅠ and XholⅠ of the pCAGGS expression vector (provided by Suzhou Jinweizhi Company) through the EcoRⅠ and XholⅠ restriction sites, and the recombinant expression plasmids were obtained after sequencing verification. According to the different inserted sequences, they were named pCAGGS-BF.7-S-del18, pCAGGS-BA.4.6-S-del18, pCAGGS-BQ.1-S-del18, pCAGGS-XBB.1.5-S-del18, and pCAGGS-CH.1.1-S-del18.

[0100] 2. Packaging of SARS-CoV-2 prototype strains and variant strains of pseudovirus

[0101] (1) Cell preparation: Add double antibodies (penicillin and streptomycin) and 10% FBS (fetal bovine serum) to DMEM culture medium to prepare maintenance medium. Plate HEK293T cells in a 10 cm cell culture dish and incubate at 37°C and 5% CO 2 The cells were cultured in an incubator for 12-24 hours, and the cell confluence density reached about 70-80% on the next day.

[0102] (2) Transfection: Discard the culture medium in the 10 cm culture dish and replace it with an antibiotic-free and serum-free culture medium. Take the expression plasmids of each truncated S protein obtained in step 1 above, and transfect 30 μg of plasmid / 10 cm cell culture dish with PEI. Mix the target plasmid and PEI at a ratio of 1:3 and then transfect. After 4-6 hours, replace the culture medium (DMEM culture medium containing 10% FBS and double antibodies). Incubate at 37°C and 5% CO 2 Culture in an incubator for 24 h.

[0103] (3) Adding poison: Add 500 μL (titer 10) of VSV-ΔG-EGFP (product of Wuhan Shumi Brain Science and Technology Co., Ltd., catalog number V03001) required for pseudovirus packaging. 8 PFU / 100 μL) to the HEK293T cells after transfection and incubate at 37°C, 5% CO 2 After incubation in the incubator for 2 h, the supernatant was discarded and the cells were washed once with PBS, and the culture medium (10% FBS DMEM medium and VSV-G antibody, hybridoma cells expressing the antibody were purchased from ATCC cell bank, CRL2700 TM , in order to neutralize the original VSV pseudovirus), at 37°C, 5% CO 2 Continue culturing in the incubator for 24-30 hours.

[0104] (4) Collection of virus: Centrifuge at 3000 rpm for 10 min to collect the cell supernatant, filter through a 0.45 μm sterile filter in a biosafety culture cabinet to remove cell debris, and then aliquot and store in a -80°C refrigerator.

[0105] After the above steps, pseudoviruses of SARS-CoV-2 variant strains Omicron (BF.7), Omicron (BA.4.6), Omicron (BQ.1), Omicron (XBB.1.5) and Omicron (CH.1.1) were obtained respectively.

[0106] Example 5. Evaluation of the inhibitory effect of Affibody peptides on SARS-CoV-2 prototype strains and variant strains of pseudovirus

[0107] The purpose of this example is to determine the pseudovirus inhibition effect of the polypeptide S-2 prepared in Example 2 and the derivative polypeptides of S-2 (S-2-dimer, S-2-Fc-1, S-2-Fc-2) on the prototype strain (SARS-CoV-2-PT) and the pseudovirus inhibition effect of S-2-Fc-2 on mutant strains (Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron (BA.1), Omicron (BA.2), Omicron (BA.2.12.1), Omicron (BA.2.75), Omicron (BA.4 / 5), Omicron (BF.7), Omicron (BA.4.6), Omicron (BQ.1), Omicron (BQ.1.1), Omicron (XBB), Omicron (XBB.1.5) and Omicron (CH.1.1)).

[0108] Experimental groups: Vero-E6 cells (from ATCC cell bank) + DMEM medium (blank control group, cells were not infected with the virus); Vero-E6 cells + SARS-CoV-2 prototype strain or variant strain pseudovirus + DMEM medium (negative control group, cells were infected with the virus but not treated with polypeptides); Vero-E6 cells + SARS-CoV-2 prototype strain or variant strain pseudovirus + polypeptides (S-2 and S-2 derivative polypeptides (S-2-dimer, S-2-Fc-1, S-2-Fc-2)) (experimental group, cells were infected with the virus and treated with polypeptides).

[0109] Preparation of peptide gradient dilution solution: Add double antibodies (penicillin and streptomycin) and 10% FBS to DMEM culture medium to prepare maintenance solution, and use this maintenance solution to dilute the purified S-2 peptide 2-fold gradient stepwise to different concentrations (7.14, 3.57, 1.79, 0.89, 0.45, 0.22, 0.11, 0.06 μM) of the test solution, and the S-2-dimer peptide 4-fold gradient stepwise to different concentrations (3571.43, 892.86, 223.21, 55.80, 13.95, 3.49, 0.87, 0.2 2nM) test solution, S-2-Fc-1 polypeptide was diluted stepwise with a gradient of 3 times to test solutions of different concentrations (781.25, 390.63, 195.31, 97.66, 48.83, 24.41, 12.21, 6.10nM), S-2-Fc-2 polypeptide was diluted stepwise with a gradient of 3 times to test solutions of different concentrations (2500.00, 833.33, 277.78, 92.59, 30.86, 10.29, 3.43, 1.14, 0.38, 0.13nM), and the volume of each concentration was 50μL.

[0110] Determination of pseudovirus dosage: The SARS-CoV-2 prototype strain or variant strain pseudovirus was diluted stepwise with maintenance solution in a 3-10-fold gradient, quantified on Vero-E6 cells, and the dilution at 1000 PFU / 50 μL was used as the virus dosage for evaluating the inhibitory effect of the polypeptide.

[0111] The method for determining the virus inhibition effect is:

[0112] a) Add double antibodies (penicillin and streptomycin) and 10% FBS to DMEM culture medium to prepare a maintenance solution, and use the maintenance solution to dilute the SARS-CoV-2 prototype strain or variant strain pseudovirus to a titer of 1000 PFU / 50 μL.

[0113] b) Dilute the peptides in the experimental group in the above method with the maintenance solution, each concentration volume is 50 μL, and add 50 μL of the diluted SARS-CoV-2 prototype strain or variant strain pseudovirus, and place the mixture at 37°C and 5% CO 2 Incubate in incubator for 1h.

[0114] c) Vero-E6 cells were seeded in 96-well plates and placed at 37°C with 5% CO 2 Incubate in an incubator overnight, and when the cells grow to 90%-100% density, discard the culture supernatant.

[0115] d) Add the polypeptide-pseudovirus mixture in step b) to the 96-well plate in step c) (100 μL / well) and place at 37°C, 5% CO 2 Incubate in incubator for 15h.

[0116] e) The infected cells express EGFP protein (the successfully prepared pseudovirus expresses EGFP protein after infecting cells because its genome contains the EGFP gene, showing green fluorescence. The pseudovirus neutralized by the drug cannot invade the cells and does not produce green fluorescence. The detailed principle reference is as follows: Nie J, Li Q, Wu J, et al. Quantification of SARS-CoV-2neutralizing antibody by a pseudotyped virus-based assay. Nat Protoc. 2020). The green fluorescence number, that is, the number of infected cells, is calculated using a high-content cell imaging analysis system. The inhibition rate = (1-(fluorescence number of the experimental group-fluorescence number of the blank control group) / (fluorescence number of the negative control group-fluorescence number of the blank control group))*100(%). The IC of each polypeptide was calculated using GraphPad 50 .

[0117] The results are as follows Figure 7 , Figure 8 As shown in Table 1. It can be seen that the Affibody peptide S-2-Fc-2 has good broad-spectrum neutralizing activity against the prototype and mutant pseudoviruses of SARS-CoV-2.

[0118] Table 1. Evaluation of the inhibitory effect of the derivative peptide S-2-Fc-2 of Affibody peptide S-2 on the SARS-CoV-2 prototype strain and variant strain pseudovirus

[0119]

[0120] Note: The results in the table are IC 50 In the table, "PC\T\" represents the SARS-CoV-2 prototype strain pseudovirus.

[0121] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A peptide capable of inhibiting SARS-CoV-2, Features: The amino acid sequence of the polypeptide is shown in SEQ ID No.

1.

2. A polypeptide polymer capable of inhibiting SARS-CoV-2, Features: The polypeptide multimer is a multimer formed by connecting two or more polypeptides according to claim 1 via a flexible peptide.

3. The polypeptide multimer according to claim 2, Features: The amino acid sequence of the polypeptide polymer is shown in SEQ ID No.

3.

4. A fusion protein capable of inhibiting SARS-CoV-2, which is any of the following: (A1) is formed by fusion of the polypeptide according to claim 1 and an Fc fragment and / or a tag protein and / or other functional protein connected to the amino terminus or carboxyl terminus of the polypeptide; (A2) is formed by fusing the polypeptide multimer according to claim 2 with an Fc fragment and / or a tag protein or other functional protein connected to the amino terminus or carboxyl terminus of the polypeptide multimer.

5. The fusion protein according to claim 4, Features: The amino acid sequence of the fusion protein is shown as SEQ ID No.2 or SEQ ID No.

4.

6. A nucleic acid molecule encoding the polypeptide according to claim 1, the polypeptide multimer according to claim 2 or 3, or the fusion protein according to claim 4 or 5.

7. The nucleic acid molecule according to claim 6, Features: The nucleic acid molecule encoding the polypeptide of claim 1 is shown in SEQ ID No. 5; or The nucleic acid molecule encoding the polypeptide multimer of claim 3 is shown in SEQ ID No. 7; or The nucleic acid molecule encoding the fusion protein shown in SEQ ID No. 2 of claim 5 is shown in SEQ ID No. 6; or The nucleic acid molecule encoding the fusion protein shown in SEQ ID No. 4 in claim 5 is shown in SEQ ID No.

8.

8. An expression cassette or recombinant vector or recombinant microorganism or transgenic cell line containing the nucleic acid molecule according to claim 6 or 7.

9. A connector formed by covalently or non-covalently linking the polypeptide according to claim 1, the polypeptide multimer according to claim 2 or 3, or the fusion protein according to claim 4 or 5 with a fluorescent group or a radioactive group or biotin or streptomycin or a nanomaterial.

10. Any of the following applications: (B1) Use of the polypeptide according to claim 1, the polypeptide multimer according to claim 2 or 3, the fusion protein according to claim 4 or 5, the nucleic acid molecule according to claim 6 or 7, the expression cassette or recombinant vector or recombinant microorganism or transgenic cell line according to claim 8, or the connector according to claim 9 in the preparation of a product for inhibiting SARS-CoV-2; (B2) Use of the polypeptide according to claim 1, the polypeptide multimer according to claim 2 or 3, the fusion protein according to claim 4 or 5, the nucleic acid molecule according to claim 6 or 7, the expression cassette or recombinant vector or recombinant microorganism or transgenic cell line according to claim 8, or the connector according to claim 9 in the preparation of a product for treating and / or preventing diseases caused by SARS-CoV-2 infection; (B3) Use of the polypeptide according to claim 1, the polypeptide multimer according to claim 2 or 3, the fusion protein according to claim 4 or 5, the nucleic acid molecule according to claim 6 or 7, the expression cassette or recombinant vector or recombinant microorganism or transgenic cell line according to claim 8, or the connector according to claim 9 in the preparation of a product for improving symptoms caused by SARS-CoV-2 infection; (B4) Use of the polypeptide according to claim 1, the polypeptide multimer according to claim 2 or 3, the fusion protein according to claim 4 or 5, the nucleic acid molecule according to claim 6 or 7, the expression cassette or recombinant vector or recombinant microorganism or transgenic cell line according to claim 8, or the connector according to claim 9 in the preparation of a product capable of binding to the S protein of SARS-CoV-2; (B5) Use of the polypeptide according to claim 1, the polypeptide multimer according to claim 2 or 3, the fusion protein according to claim 4 or 5, the nucleic acid molecule according to claim 6 or 7, the expression cassette or recombinant vector or recombinant microorganism or transgenic cell line according to claim 8, or the connector according to claim 9 in the preparation of a product for diagnosing a disease caused by SARS-CoV-2 infection; Furthermore, the SARS-CoV-2 is a prototype strain or a variant strain.

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