Construction method and application of small peptide combined with SARS-CoV-2 receptor structural domain

Abstract

The invention discloses a construction method of a small peptide combined with an SARS-CoV-2 receptor structural domain. The construction method comprises the following steps: designing an SARS-CoV-2 virus SPIKE protein RBD domain sequence; the method comprises the following steps: carrying out RBD domain cloning on the SPIKE protein of the SARS-CoV-2 virus; constructing a yeast two-hybrid Bit strain; performing yeast two-hybrid screening; and analyzing the target fragment sequence. The method has the beneficial effects that the method has high diversity; the sensitivity is high; the flux is high; soluble expression is realized; the polypeptide has modifiability, and the potential of the polypeptide as a vaccine or a drug is enhanced; the compound can be applied to research and development of antiviral drugs, has high affinity and specificity, and can be combined with RBD and block viruses from entering cells; the compound can be used as an auxiliary agent of vaccines and is used for enhancing immunogenicity and protective effect of the vaccines. The kit can be used for diagnosis and detection of viruses, can be combined with RBD to form a specific compound, and can be used as a recognition and detection tool in virus detection.

Description

Technical Field

[0001] The present invention relates to a construction method and application of a small peptide that binds to a SARS-CoV-2 receptor domain, and belongs to the technical field of bioengineering. Background Art

[0002] SARS-CoV-2 is a virus belonging to the Coronaviridae family, and it is the pathogen that causes COVID-19. SARS-CoV-2 is an enveloped virus with a positive-strand RNA genome. Its genome is about 30,000 nucleotides long and encodes multiple proteins, including spike protein (S protein), envelope protein (E protein), nucleocapsid protein (M protein), and nucleocapsid protein (N protein). SARS-CoV-2 is mainly transmitted through droplets, and the virus can enter the nose, mouth, or eyes of another person through respiratory secretions when an infected person coughs, sneezes, or talks. In addition, the virus can also be transmitted by touching contaminated surfaces and then touching the mouth, nose, or eyes. SARS-CoV-2 was initially thought to have been transmitted to humans from wild animals, but the exact host remains undetermined. Studies have shown that bats may be the natural host of the virus, but the intermediate host is still under investigation. Like other RNA viruses, SARS-CoV-2 also has the ability to mutate. Over time, the virus may have some variants, some of which may have different transmissibility, severity, or immune escape capabilities. This emphasizes the importance of monitoring viral mutations and ongoing research. Vaccines against SARS-CoV-2 have been developed and are available for use. The goal of the vaccine is to activate the human immune system so that it produces an immune response to the virus. In addition, scientists are also researching and developing antiviral drugs and other treatments to reduce the symptoms and severity of COVID-19. Overall, SARS-CoV-2 is the coronavirus that causes COVID-19, which has had a significant impact on global health and society. Studying and understanding the characteristics of this virus is critical to controlling and preventing the spread of COVID-19.

[0003] The SARS-CoV-2 receptor binding domain (RBD) is an important region on the viral spike protein (S protein) that binds to receptors on host cells and mediates the virus's entry into cells. The SARS-CoV-2 spike protein is a surface protein of the virus that is present on the virus's outer shell and forms the crown-like shape of the virus, hence its name. The spike protein consists of two subunits (S1 and S2), of which the S1 subunit contains the RBD. The RBD is located at the N-terminus of the S1 subunit and is a key site for interaction with receptors on host cells. For SARS-CoV-2, the RBD mainly binds to the ACE2 (angiotensin-converting enzyme 2) receptor on host cells to form a binding bridge between the virus and the cell. This binding enables the virus to enter host cells, infect and replicate itself. The structure of the RBD is highly flexible and specific. It achieves the binding between the virus and the host cell by interacting with the ACE2 receptor, a process that is a key step in the pathogenesis of COVID-19. Studying the structure and function of the RBD can help scientists understand how the virus enters host cells, thereby providing important clues for the design of antiviral drugs and vaccine development. Using techniques such as X-ray crystallography and electron microscopy, the researchers successfully resolved the high-resolution structure of the SARS-CoV-2 spike protein and RBD, which provides a basis for understanding the virus's infection mechanism and developing vaccines and therapeutic drugs targeting RBD. As the key connection point between the SARS-CoV-2 virus and the host cell, RBD is of great significance in the study and response to COVID-19.

[0004] The binding of the SARS-CoV-2 receptor binding domain (RBD) to the ACE2 receptor on the host cell is a key step for the virus to enter the cell. Due to the importance of the interaction between RBD and ACE2 to viral invasion, it is worth studying how to construct small peptides that can bind to RBD to interfere with the binding of the virus to the host cell, thereby preventing the virus from entering the cell. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for constructing and applying a small peptide that binds to the SARS-CoV-2 receptor domain. The small peptide has high affinity and specificity, can bind to RBD and block the virus from entering cells.

[0006] The present invention is achieved through the following scheme: a small peptide that binds to the SARS-CoV-2 receptor domain, and its sequence is: WGPMLWRAFDRAMSAGLGGW.

[0007] A small peptide method that binds to the SARS-CoV-2 receptor domain, whose sequence is one of the following 19:

[0008] Peptide1 PPSTRVPVTTFVGTELGSTA

[0009] Peptide2 WGPMLWRAFDRAMSAGLGGW

[0010] Peptide4 VRAMRLMQWLLCVLSTAARS

[0011] Peptide5 LPLSSEVGASTGTYRLRLLL

[0012] Peptide7 SRFASTTFSPRLVGVAAACQ

[0013] Peptide9 IGRAAPLSLLLFWALTKEPK

[0014] Peptide13 LAPTATLASSVAIHLWKGLI

[0015] Peptide15 PWSFFFCVSGRLCRLRVCGA

[0016] Peptide16 LRTLSCRLLVALMQFASAEC

[0017] Peptide17 CPFARRPWFPMSLRLVLVCF

[0018] Peptide18 PSSSMFRTELTAGGSL

[0019] Peptide19 LVQLMWPAIRCSRGFSGAYV

[0020] Peptide21 LVQLMWPAIRCSRGFRVRTF

[0021] Peptide22 LLFFVLPLRLPREVTRVSPA

[0022] Peptide23 SRFPLSLYVTLHTATLGRMR

[0023] Peptide26 YTTLPRRLPRGRPAPPRSIG

[0024] Peptide28 GGQDLSLTSVSLLARFLLPW

[0025] Peptide29 TSGCPRSWPVGWSTLAFTGL Peptide32 WQSRCLPGALHLVLFLSVAK.

[0026] A method for constructing a small peptide that binds to a SARS-CoV-2 receptor domain comprises the following steps:

[0027] Step 1: Design of the RBD domain sequence of the SARS-CoV-2 virus SPIKE protein;

[0028] Step 2: Cloning of the RBD domain of the SARS-CoV-2 virus SPIKE protein;

[0029] Step 3: Construction of yeast two-hybrid Bait strain;

[0030] Step 4: Yeast two-hybrid screening;

[0031] Step 5: Analysis of target fragment sequence.

[0032] The step 1 includes the following processes: selecting a spike sequence, removing a transmembrane structure sequence, a codon-optimized DNA sequence, adding restriction sites, and sending a synthetic sequence.

[0033] The step 2 includes the following processes: sequence synthesis, fragment amplification, enzyme digestion recovery, ligation reaction, Escherichia coli transformation, and plasmid extraction.

[0034] The amplification primers used for the fragment amplification are:

[0035] Primer1 is GAGGCCGAATTCAGAGTCCAACCAACTGAGAGT,

[0036] Primer2 is GTCGACGGATCCTAGAAATTTACACACTTGTTC.

[0037] The step three includes AH109 competent cell preparation and AH109 transformation of Bait plasmid.

[0038] The step 4 includes self-activation detection and library screening.

[0039] The step five includes PCR amplification of the target fragment, sequencing of the PCR product of the target fragment, and conserved sequence of the SPIKE-RBD binding peptide. The PCR amplification of the target fragment uses universal primers of the pGADT7 vector to amplify the inserted fragments of the target plasmid in all positive clone strains, and the amplified products that meet the library gene length are screened by agarose gel electrophoresis, and the amplified products are mixed in equal amounts and all gene fragments in the PCR products are recovered using a magnetic bead recovery method. The PCR product sequencing of the target fragment is to amplify the 36 positive clones finally obtained with AD-REV and AD-FWD primers to obtain 35 PCR fragments, and then obtain 34 random peptide library sequences by Sanger sequencing.

[0040] The application of a small peptide that binds to the SARS-CoV-2 receptor domain is used to block the binding between the virus and the host cell and inhibit the virus from entering the cell.

[0041] The beneficial effects of the present invention are:

[0042] 1. The present invention has high diversity: the random peptide yeast library contains a large number of different random peptide sequences, which can cover a wide range of structural and functional spaces; it can screen out binding proteins with high diversity and discover candidate proteins with high affinity and high specificity;

[0043] 2. The present invention has high sensitivity: Due to the high efficiency and controllability of the yeast expression system, sensitive screening of low-concentration target proteins (such as SARS-CoV-2 RBD) can be achieved; this enables the detection of candidate proteins with low binding affinity but still potential therapeutic effects;

[0044] 3. The present invention has high throughput: the yeast expression system can perform high-throughput screening, can process a large number of yeast clones at the same time, can screen a large number of binding proteins in a relatively short time, and improve the efficiency of discovering potential drug candidates;

[0045] 4. The present invention has soluble expression: the yeast expression system can express the candidate protein in a soluble state, which is very important for subsequent structural and functional studies. The soluble expressed protein is easy to purify and further characterize, which helps to determine its mechanism of action in the interaction with SARS-CoV-2 RBD;

[0046] 5. The present invention is transformable: by introducing genetic engineering techniques of other yeast expression systems, such as DNA recombination or exchange, the screened binding proteins can be transformed and optimized, which makes it possible to improve their affinity, specificity and stability through further engineering, thereby enhancing their potential as vaccines or drugs;

[0047] 6. The present invention can be applied to the research and development of antiviral drugs: RBD-binding peptides can be used as candidates for antiviral drugs to block the binding between viruses and host cells, thereby inhibiting the entry of viruses into cells. These peptides can interact with RBD to compete for the binding sites of ACE2 and RBD, thereby blocking the invasion of viruses;

[0048] 7. The present invention can be applied to vaccine adjuvants: RBD-binding peptides can be used as vaccine adjuvants to enhance the immunogenicity and protective effect of vaccines. These peptides can bind to viral proteins in vaccines and activate the immune system to produce antibodies and cellular immune responses against RBD. By introducing RBD-binding peptides, vaccines can better simulate the interaction between viruses and host cells and improve the efficacy and durability of immune responses.

[0049] 8. The present invention can be applied to diagnostic tools: RBD-binding peptides can be used for virus diagnosis and detection. These peptides can bind to RBD and form specific complexes, and serve as identification and detection tools in virus detection. This method can be used to quickly, sensitively and specifically detect SARS-CoV-2 infection, and help track and monitor the virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a positive clone of AH109 transformed with pGBKT7-SPIKE_RBD.

[0051] Figure 2 Screening process of SPIKE-RBD binding peptides by yeast two-hybridization: (A) Microscope image of the mating process of AH109 and Y187, (B) Positive hybridizing yeast clones grown on -Trp / -Leu / -His / -Ade SD medium.

[0052] Figure 3 For the multiple sequence alignment of 34 peptides interacting with Spike, the quality score of each site in the alignment was calculated by summing, and a consensus sequence was generated based on the alignment analysis. DETAILED DESCRIPTION

[0053] Combine the following Figure 1-3 The present invention is further described, but the protection scope of the present invention is not limited to the described contents.

[0054] For the sake of clarity, not all features of an actual embodiment are described. In the following description, well-known functions and structures are not described in detail because they would obscure the invention with unnecessary detail. It should be recognized that in the development of any actual embodiment, a large number of implementation details must be made to achieve the specific goals of the developer, such as changing from one embodiment to another according to relevant system or relevant business limitations. In addition, it should be recognized that such development work may be complex and time-consuming, but it is merely routine work for those skilled in the art.

[0055] A small peptide that binds to the SARS-CoV-2 receptor domain, whose sequence is: WGPMLWRAFDRAMSAGLGGW.

[0056] A small peptide that binds to the SARS-CoV-2 receptor domain, whose sequence is one of the following 19:

[0057] Peptide1 PPSTRVPVTTFVGTELGSTA

[0058] Peptide2 WGPMLWRAFDRAMSAGLGGW

[0059] Peptide4 VRAMRLMQWLLCVLSTAARS

[0060] Peptide5 LPLSSEVGASTGTYRLRLLL

[0061] Peptide7 SRFASTTFSPRLVGVAAACQ

[0062] Peptide9 IGRAAPLSLLLFWALTKEPK

[0063] Peptide13 LAPTATLASSVAIHLWKGLI

[0064] Peptide15 PWSFFFCVSGLCRLLRVCGA

[0065] Peptide16 LRTLSCRLLVALMQFASAEC

[0066] Peptide17 CPFARRPWFPMSLRLVLVCF

[0067] Peptide18 PSSSMFRTELTAGGSL

[0068] Peptide19 LVQLMWPAIRCSRGFSGAYV

[0069] Peptide21LVQLMWPAIRCSRGFRVRTF

[0070] Peptide22 LLFFVLPLRLPREVTRVSPA

[0071] Peptide23 SRFPLSLYVTLHTATLGRMR

[0072] Peptide26 YTTLPRRLPRGRPAPPRSIG

[0073] Peptide28 GGQDLSLTSVSLLARFLLPW

[0074] Peptide29 TSGCPRSWPVGWSTLAFTGL

[0075] Peptide32 WQSRCLPGALHLVLFLSVAK.

[0076] A method for constructing a small peptide that binds to a SARS-CoV-2 receptor domain comprises the following steps:

[0077] Step 1. Design of the RBD domain sequence of the SARS-CoV-2 virus SPIKE protein: This includes the following processes: selecting the spike sequence, removing the transmembrane structure sequence, codon-optimized DNA sequence, adding restriction sites, and sending the synthetic sequence.

[0078] Step 2: Cloning of the RBD domain of the SARS-CoV-2 virus SPIKE protein: including the following processes: sequence synthesis, fragment amplification, enzyme digestion recovery, ligation reaction, E. coli transformation, plasmid extraction, and amplification primers used for fragment amplification:

[0079] Primer1 is GAGGCCGAATTCAGAGTCCAACCAACTGAGAGT,

[0080] Primer2 is GTCGACGGATCCTAGAAATTTACACACTTGTTC.

[0081] Step 3: Construction of yeast two-hybrid Bait strain: including preparation of AH109 competent cells and transformation of AH109 with Bait plasmid;

[0082] Step 4: Yeast two-hybrid screening: including self-activation detection and library screening.

[0083] Step 5, analysis of the target fragment sequence: including PCR amplification of the target fragment, sequencing of the PCR product of the target fragment, and conserved sequence of the SPIKE-RBD binding peptide. The PCR amplification of the target fragment uses the universal primers of the pGADT7 vector to amplify the insert fragment of the target plasmid in all positive clone strains, and the amplification products that meet the library gene length are screened by agarose gel electrophoresis, and the amplification products are mixed in equal amounts and all gene fragments in the PCR products are recovered using a magnetic bead recovery method. The PCR product sequencing of the target fragment is to amplify the 36 positive clones finally obtained with AD-REV and AD-FWD primers to obtain 35 PCR fragments, and then obtain 34 random peptide library sequences by Sanger sequencing.

[0084] The application of a small peptide that binds to the SARS-CoV-2 receptor domain is used to block the binding between the virus and the host cell and inhibit the virus from entering the cell.

[0085] A small peptide that binds to the SARS-CoV-2 receptor domain as a vaccine adjuvant.

[0086] Application of a small peptide binding to the SARS-CoV-2 receptor domain in virus diagnosis and detection.

[0087] Example:

[0088] Step 1: Design of the RBD domain sequence of the SARS-CoV-2 virus SPIKE protein.

[0089] 1. Select the spike sequence

[0090] The protein name YP_009724390.1 surface glycoprotein [Severe acute respiratory syndrome coronavirus 2] was changed to Spro

[0091] >Spro_1273aa

[0092] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0093] 2. Remove the transmembrane domain sequence

[0094] >RBD_319-541_(223aa)

[0095] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN

[0096] 3. DNA sequence after codon optimization

[0097] >Spo_RBD_DNA sequences(Modified according to Yeast Condon preference,669nt)

[0098] AGAGTCCAACCAACTGAGAGTATAGTTCGTTTTCCTAACATCACTAACCTTTGTCCTTTTGGCGAAGTTTTTAATGCTACTCGTTTTGCTTCCGTATATGCTTGGAATCGTAAAAGAATTTCTAATTGTGTTGCAGATTATTCAGTTCTTTATAATTCTGCTTCATTTTCTACTTTTAAGTGCTATGGTGTTTCTCCTACTAAATTGAATGATCTTTGTTTTACAAATGTTTACGCTGACAGTTTCGTCATCAGAGGCGACGAAGTTCGTCAAATAGCTCCTGGTCAAACTGGAAAAATTGCTGATTACAACTATAAATTACCAGATGACTTCACTGGTTGTGTCATAGCTTGGAACTCCAACAACTTGGATTCTAAGGTTGGCGGTAATTACAATTACTTATATCGTTTATTCAGAAAATCAAACTTAAAGCCCTTTGAACGTGATATTTCCACCGAAATTTATCAGGCTGGAAGTACGCCTTGCAATGGTGTAGAGGGCTTCAATTGCTATTTCCCATTGCAATCATATGGTTTTCAACCAACTAATGGTGTAGGTTACCAACCTTATAGAGTTGTTGTTTTATCTTTTGAGTTGTTACATGCTCCTGCAACTGTATGTGGTCCTAAGAAAAGTACCAATTTAGTTAAGAACAAGTGTGTAAATTTC

[0099] 4. Add restriction enzyme sites (ECORI + BamHI)

[0100] ECOR1: GAATTC

[0101] BamH1: GGATCC

[0102] Stop Codon: TAG

[0103] >Spro_RBD_DNA_synthesis(EcoRI - Apro - stop codon - BamH1)

[0104] GAATTCAGAGTCCAACCAACTGAGAGTATAGTTCGTTTTCCTAACATCACTAACCTTTGTCCTTTTGGCGAAGTTTTTAATGCTACTCGTTTTGCTTCCGTATATGCTTGGAATCGTAAAAGAATTTCTAATTGTGTTGCAGATTATTCAGTTCTTTATAATTCTGCTTCATTTTCTACTTTTAAGTGCTATGGTGTTTCTCCTACTAAATTGAATGATCTTTGTTTTACAAATGTTTACGCTGACAGTTTCGTCATCAGAGGCGACGAAGTTCGTCAAATAGCTCCTGGTCAAACTGGAAAAATTGCTGATTACAACTATAAATTACCAGATGACTTCACTGGTTGTGTCATAGCTTGGAACTCCAACAACTTGGATTCTAAGGTTGGCGGTAATTACAATTACTTATATCGTTTATTCAGAAAATCAAACTTAAAGCCCTTTGAACGTGATATTTCCACCGAAATTTATCAGGCTGGAAGTACGCCTTGCAATGGTGTAGAGGGCTTCAATTGCTATTTCCCATTGCAATCATATGGTTTTCAACCAACTAATGGTGTAGGTTACCAACCTTATAGAGTTGTTGTTTTATCTTTTGAGTTGTTACATGCTCCTGCAACTGTATGTGGTCCTAAGAAAAGTACCAATTTAGTTAAGAACAAGTGTGTAAATTTCTAGGATCC

[0105] 5. Send the synthesized sequence:

[0106] >iGEM1_Seq1_Spro_RBD

[0107] GAATTCAGAGTCCAACCAACTGAGAGTATAGTTCGTTTTCCTAACATCACTAACCTTTGTCCTTTTGGCGAAGTTTTTAATGCTACTCGTTTTGCTTCCGTATATGCTTGGAATCGTAAAAGAATTTCTAATTGTGTTGCAGATTA TTCAGTTCTTTATAATTCTGCTTCATTTTCTACTTTTAAGTGCTATGGTGTTTCTCCTACTAAATTGAATGATCTTTGTTTTACAAATGTTTACGCTGACAGTTTCGTCATCAGAGGCGACGAAGTTCGTCAAATAGCTCCTGGTCAAACTGGAAAAATTGCTGATTACAACTATAAATTACCAGATGACTTCACTGGTTGTGTCATAGCTTGGAACTCCAACAACTTGGATTCTAAGGTTGGCGGTAATTACAATTACTTATATCGTTTATTCAGAAAATCAAACTTAAAGCCCTTTGAACGTGATATTTCCACCGAAATTTATCAGGCTGGAAGTACGCCTTGCAATGGTGTAGAGGGCTTCAATTGCTATTTCCCATTGCAATCATATGGTTTTCAACCAACTAATGGTGTAGGTTACCAACCTTATAGAGTTGTTGTTTTATCTTTTGAGTTGTTACATGCTCCTGCAACTGTATGTGGTCCTAAGAAAAGTACCAATTTAGTTAAGAACAAGTGTGTAAATTTCTAGGATCC

[0108] Step 2: Cloning of the RBD domain of the SARS-CoV-2 virus SPIKE protein.

[0109] 1. Synthetic sequence:

[0110] >iGEM1_Seq1_Spro_RBD

[0111] GAATTCAGAGTCCAACCAACTGAGAGTATAGTTCGTTTTCCTAACATCACTAACCTTTGTCCTTTTGGCGAAGTTTTTAATGCTACTCGTTTTGCTTCCGTATATGCTTGGAATCGTAAAAGAATTTCTAATTGTGTTGCAGATTATTCAGTTCTTTATAATTCTGCTTCATTTTCTACTTTTAAGTGCTATGGTGTTTCTCCTACTAAATTGAATGATCTTTGTTTTACAAATGTTTACGCTGACAGTTTCGTCATCAGAGGCGACGAAGTTCGTCAAATAGCTCCTGGTCAAACTGGAAAAATTGCTGATTACAACTATAAATTACCAGATGACTTCACTGGTTGTGTCATAGCTTGGAACTCCAACAACTTGGATTCTAAGGTTGGCGGTAATTACAATTACTTATATCGTTTATTCAGAAAATCAAACTTAAAGCCCTTTGAACGTGATATTTCCACCGAAATTTATCAGGCTGGAAGTACGCCTTGCAATGGTGTAGAGGGCTTCAATTGCTATTTCCCATTGCAATCATATGGTTTTCAACCAACTAATGGTGTAGGTTACCAACCTTATAGAGTTGTTGTTTTATCTTTTGAGTTGTTACATGCTCCTGCAACTGTATGTGGTCCTAAGAAAAGTACCAATTTAGTTAAGAACAAGTGTGTAAATTTCTAGGATCC

[0112] >Primer 1GAGGCCGAATTCAGAGTCCAACCAACTGAGAGT

[0113] >Primer 2GTCGACGGATCCTAGAAATTTACACACTTGTTC

[0114] 2. Fragment amplification: Using iGEM1_Seq1_Spro_RBD as a template and Primer 1 and Primer 2 as primers, amplify the DNA fragment of SPIKE_RBD of the SARS-CoV-2 virus, labeled as Fragment RBD.

[0115] 3. Enzyme digestion recovery: EcoR1 and BamH1 were used to double-digest pGBKT7 and Fragment RBD fragments; gel excision was used to recover the vector backbone and Fragment RBD after enzyme digestion. They were recorded as pGBKT7-Digested and Fragment RBD-Digested respectively.

[0116] 4. Ligation reaction: Use T4-DNA ligase to ligate the vector backbone pGBKT7-Digested and the inserted fragment RBD-Digested after enzyme digestion according to the standard procedure.

[0117] 5. E. coli transformation: The ligation product was transformed into DH10B competent cells according to the E. coli electroporation procedure. The transformation product was spread on LB plates containing 50 mg / L karatomycin. After 18 hours, plaque PCR was used to screen positive clones.

[0118] 6. Plasmid extraction: The confirmed positive clone plaques were inoculated into 5 mL of liquid culture medium containing 50 mg / L karatomycin. After overnight culture, the plasmid was extracted and marked as pGBKT7-SPIKE_RBD.

[0119] Step 3: Construction of yeast two-hybrid Bait strain.

[0120] 1. Preparation of competent cells of AH109: Take out the AH109 cryopreserved solution from the -80℃ refrigerator, streak it on YPD medium, grow it in a 30℃ incubator, and prepare competent cells of AH109 yeast according to standard procedures for use.

[0121] 2. Preparation of competent yeast.

[0122] (1) In a clean bench, use a sterilized pipette tip to pick up a well-growing monoclonal yeast plaque and place it in 1 mL of YPDA liquid culture medium. Shake vigorously for 2-3 minutes to evenly disperse the yeast clumps in the culture medium.

[0123] (2) Add the yeast-containing culture medium to 5 mL of YPDA liquid culture medium, and culture in a 30°C incubator with shaking at 230 rpm for about 16 h. When the OD600 value of the culture medium is about 0.8, remove the culture medium and set it aside for later use.

[0124] (3) Take 2 mL of the culture medium in (2) and transfer it into 25 mL of YPDA liquid medium. Incubate the culture medium at 30°C and 230 rpm for 3-5 h. When the OD600 value is between 0.6 and 0.8, remove the culture medium for later use. Note that the OD600 value should not be too high or too low, otherwise it will affect the transformation efficiency.

[0125] (4) Add 1 mL of the above yeast solution to a 2 mL centrifuge tube and centrifuge at 700 rcf for 5 min at room temperature. Discard the supernatant.

[0126] (5) Add 500 μL of autoclaved double-distilled water to each centrifuge tube, gently blow to suspend the precipitate, centrifuge at 700 rcf for 5 min at room temperature, and discard the supernatant.

[0127] (6) Add 300 μL of 1×TE / LiAc solution to the centrifuge tube, pipette to suspend the bacterial pellet, centrifuge at 12,000 rpm for 15–30 s, and discard the supernatant.

[0128] (7) Add 60 μL of 1×TE / LiAc solution to the centrifuge tube to resuspend the bacterial pellet and place on ice for later use.

[0129] 3. AH109 transformation with Bait plasmid: Take 1 ug of pGBKT7-SPIKE_RBD plasmid and transform AH109 competent cells according to the standard yeast transformation procedure. Transform the culture dish coated with -trp SD yeast medium and grow in a 30°C incubator for 4 days. After that, the positive clones can be obtained by plaque detection (such as Figure 1 shown).

[0130] 4. Standard steps for yeast transformation.

[0131] (1) Take 1 mL of herring sperm DNA and place it in a 100°C metal bath for 5 min to denature it. Then place it on ice for 5 min. Repeat this process once and place it on ice for later use.

[0132] (2) In a clean bench, on ice, add 1 μL of the constructed plasmid DNA (about 100 ng) and 3 μL of denatured herring sperm DNA (SS-DNA) to the newly prepared yeast competent cells and mix by gently pipetting.

[0133] (3) Add 500 μL of PEG / LiAc solution, gently pipette to mix, and place in a 30°C incubator for 30 min, slowly inverting and mixing every 10 min.

[0134] (4) Add 20 μL DMSO and mix by gently pipetting. Place in a 42°C water bath for 15 min, slowly inverting and mixing every 5 min.

[0135] (5) Place the tube on ice for 1–2 min, centrifuge at 12,000 rpm for 15–30 s, and discard the supernatant.

[0136] (6) Add 1 mL of physiological saline (0.9% NaCl) to each centrifuge tube, resuspend the yeast cell pellet at the bottom of the tube, and then plate on a D / -Trp plate in a 60:600 μL ratio.

[0137] (7) Seal the coated plate with sealing film and place it upside down in a 30°C incubator for 2-4 days.

[0138] Step 4: Yeast two-hybrid screening.

[0139] 1. Self-activation detection: pGBKT7-SPIKE_RBD / pGADT7 were transformed into Y2HGold competent cells, and the positive control pGADT7-T / pGBKT7-53 and the negative control pGADT7-T / pGBKT7-Lam were transformed at the same time, and they were spread on SD / -Trp-Leu plates respectively. When a single colony grows on the SD / -Trp-Leu plate, a single colony is picked, a line is drawn on SD / -Trp-Leu-Ade-His / X, and cultured at 30°C for 3-5d to observe the growth and color development of yeast. In the present invention, pGBKT7-SPIKE_RBD has no self-activation. It can be used for subsequent yeast library screening.

[0140] 2. Library screening: Pick the positive clones of AH109 transformed with the pGBKT7-SPIKE_RBD plasmid in the above step and apply them to SD / -Trp medium, and use yeast complete medium 2xYPDA to expand the monoclonal strain; take 50ml of the expanded bait protein strain and 1ml of Y187 yeast library with 20 aa random peptides linked to AD (another patent applied by the applicant at the same time) and hybridize them at 30°C and 35rpm for 23h; take a small amount of hybridization solution and detect it under a microscope, and apply the qualified hybridization solution to SD / -Trp / -Leu / -His medium. After three days of cultivation, take the surviving strains and inoculate them in SD / -Trp / -Leu and SD / -Trp / -Leu / -His+x-а-Gal medium; after three days of cultivation, the surviving strains are the positive clone strains containing the bait protein interaction gene (such as Figure 2 shown).

[0141] Step 5: Analysis of target fragment sequence.

[0142] 1. PCR amplification of target fragments: Use universal primers of pGADT7 vector to amplify the insert fragments of target plasmids in all positive clone strains, screen the amplified products that meet the gene length of the library by agarose gel electrophoresis, mix them in equal amounts, and use magnetic bead recovery method to recover all gene fragments in the PCR products.

[0143] 2. Sequencing of PCR products of target fragments: Finally, 36 positive clones were obtained. After amplification using AD-REV and AD-FWD primers, 35 PCR fragments were obtained, and then 34 random peptide library sequences were obtained by Sanger sequencing (as shown in Table 1).

[0144] Table 1. Amino acid sequences of 34 SPIKE-RBD binding peptides

[0145]

[0146]

[0147] 3. SPIKE-RBD binding peptide conserved sequence: These 34 sequences were aligned and the conserved domains (e.g. Figure 3 As shown), WGPMLWRAFDRAMSAGLGGW, a short peptide sequence that was experimentally shown to have a strong interaction with the Spkie RBD domain.

[0148] Although the technical solutions of the present invention have been described and listed in detail, it should be understood that it is obvious to those skilled in the art to make modifications to the above embodiments or adopt equivalent alternatives. These modifications or improvements made without departing from the spirit of the present invention are within the scope of protection required by the present invention.

Claims

1. A small peptide that binds to the SARS-CoV-2 receptor domain, characterized in that: The sequence is: WGPMLWRAFDRAMSAGLGGW.

2. A small peptide that binds to the SARS-CoV-2 receptor domain, characterized in that: Its sequence is one of the following 19 items: Peptide1 PPSTRVPVTTFVGTELGSTA Peptide2 WGPMLWRAFDRAMSAGLGGW Peptide4 VRAMRLMQWLLCVLSTAARS Peptide5 LPLSSEVGASTGTYRLRLLL Peptide7 SRFASTTFSPRLVGVAAACQ Peptide9 IGRAAPLSLLLFWALTKEPK Peptide13 LAPTATLASSVAIHLWKGLI Peptide15 PWSFFFCVSGLCRLLRVCGA Peptide16 LRTLSCRLLVALMQFASAEC Peptide17 CPFARRPWFPMSLRLVLVCF Peptide18 PSSSMFRTELTAGGSL Peptide19 LVQLMWPAIRCSRGFSGAYV Peptide21LVQLMWPAIRCSRGFRVRTF Peptide22 LLFFVLPLRLPREVTRVSPA Peptide23 SRFPLSLYVTLHTATLGRMR Peptide26 YTTLPRRLPRGRPAPPRSIG Peptide28 GGQDLSLTSVSLLARFLLPW Peptide29 TSGCPRSWPVGWSTLAFTGL Peptide32 WQSRCLPGALHLVLFLSVAK.

3. A method for constructing a small peptide that binds to a SARS-CoV-2 receptor domain, characterized in that: The following steps are involved: Step 1: Design of the RBD domain sequence of the SARS-CoV-2 virus SPIKE protein; Step 2: Cloning of the RBD domain of the SARS-CoV-2 virus SPIKE protein; Step 3: Construction of yeast two-hybrid Bait strain; Step 4: Yeast two-hybrid screening; Step 5: Analysis of target fragment sequence.

4. The method for constructing a small peptide that binds to a SARS-CoV-2 receptor domain according to claim 3, characterized in that: The step 1 includes the following processes: selecting a spike sequence, removing a transmembrane structure sequence, a codon-optimized DNA sequence, adding restriction sites, and sending a synthetic sequence.

5. The method for constructing a small peptide that binds to a SARS-CoV-2 receptor domain according to claim 3, characterized in that: Step 2 The process includes: sequence synthesis, fragment amplification, enzyme digestion recovery, ligation reaction, E. coli transformation, and plasmid extraction.

6. A method for constructing a small peptide that binds to a SARS-CoV-2 receptor domain according to claim 4, characterized in that: The amplification primers used for the fragment amplification are: Primer1 is GAGGCCGAATTCAGAGTCCAACCAACTGAGAGT, Primer2 is GTCGACGGATCCTAGAAATTTACACACTTGTTC.

7. The method for constructing a small peptide that binds to a SARS-CoV-2 receptor domain according to claim 3, characterized in that: The step three includes AH109 competent cell preparation and AH109 transformation of Bait plasmid.

8. The method for constructing a small peptide that binds to a SARS-CoV-2 receptor domain according to claim 3, characterized in that: The step 4 includes self-activation detection and library screening.

9. The method for constructing a small peptide that binds to a SARS-CoV-2 receptor domain according to claim 3, characterized in that: The step five includes PCR amplification of the target fragment, sequencing of the PCR product of the target fragment, and conserved sequence of the SPIKE-RBD binding peptide. The PCR amplification of the target fragment uses universal primers of the pGADT7 vector to amplify the inserted fragments of the target plasmid in all positive clone strains, and the amplified products that meet the library gene length are screened by agarose gel electrophoresis, and the amplified products are mixed in equal amounts and all gene fragments in the PCR products are recovered using a magnetic bead recovery method. The PCR product sequencing of the target fragment is to amplify the 36 positive clones finally obtained with AD-REV and AD-FWD primers to obtain 35 PCR fragments, and then obtain 34 random peptide library sequences by Sanger sequencing.

10. An application of a small peptide that binds to a SARS-CoV-2 receptor domain, characterized in that: It is used to block the binding between viruses and host cells and inhibit the virus from entering cells.

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