Mutant of RSV (Respiratory Syndrome Virus) pre-fusion conformation F protein and application thereof

Abstract

The invention relates to a mutant of an RSV pre-fusion conformation F protein, the mutant comprises amino acid mutation relative to an amino acid sequence of a wild type RSV F protein, the amino acid mutation is that amino acids at positions 392 and 493 or near the 493 are substituted by C, and a disulfide bond is formed between mutated positions 392C and 493C or near the 493C. The invention also provides a related DNA (deoxyribonucleic acid) coding sequence, a recombinant plasmid, an mRNA (messenger ribonucleic acid) sequence and a corresponding RSV vaccine. By introducing disulfide bonds at proper positions and combining with cavity filling, electrostatic mutation, proline mutation and the like, the expression and stability of the F protein with conformations before and after fusion and trimerization conformations are remarkably improved, and the F protein is remarkably inhibited from being converted into conformations after fusion. The mutant is also applied to preparation of RSV vaccines and detection of respiratory syncytial virus infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a mutant of a pre-fusion conformation of a respiratory syncytial virus F protein and its application in the development of a respiratory syncytial virus vaccine and the diagnosis of respiratory syncytial virus infection. Background Art

[0002] Respiratory Syncytial Virus (RSV) infection causes substantial morbidity and mortality in infants, young children, and the elderly, with an estimated 64 million infections and 160,000 deaths worldwide each year. Pneumonia caused by RSV infection is the leading cause of clinical mortality. To date, passive prevention with monoclonal antibodies has been limited to high-risk infants in part due to its modest efficacy. There is currently no approved safe and effective vaccine for RSV infection in my country, highlighting the urgent need for an RSV vaccine that provides a protective immune response.

[0003] The RSV genome is about 15Kb in length, containing 10 genes, encoding 11 proteins, including 8 structural proteins (F, G, M2-1, M2-2, SH, N, P, L) and 3 non-structural proteins (NS1, NS2, NS3). Among them, fusion glycoprotein (F) and attachment glycoprotein (G) are the two main envelope glycoproteins. F protein belongs to type I viral fusion protein, which mediates the membrane fusion of virus and host cell, so that the viral envelope and host cell membrane fuse to form a multi-syncytium. Neutralizing antibodies against F protein can effectively inhibit RSV virus infection. Neutralizing antibodies induced by natural RSV infection in humans are mainly against F protein; the amino acid sequence of F protein is more than 93% consistent between RSV subtypes, and its genetic stability is much higher than that of G protein. In addition, F protein is an important target for inducing the body to produce CD8+T cells. Therefore, RSV F protein is an important target for the development of RSV vaccine.

[0004] The F protein of RSV is a trimer structure, which mediates the fusion of the viral envelope and the host cell through conformational changes. Before the fusion is initiated, the F protein adopts a pre-fusion conformation, which is unstable and has a low energy barrier. During the RSV virus infection of host cells, the F protein conformation changes from the pre-fusion conformation to the post-fusion conformation, mediating the fusion of the virus and the host cell membrane. The F protein in the post-fusion conformation is very stable. The F protein in the pre-fusion conformation is very different from the F protein in the post-fusion conformation and has different antigenic epitopes. The pre-fusion conformation of the F protein has key neutralizing epitopes for RSV infection. When the F protein changes to the post-fusion conformation, most of the key neutralizing epitopes are lost. Therefore, it is crucial to maintain the stability of the trimer conformation of the F protein before fusion.

[0005] In order to solve the problem of unstable structure of pre-fusion F protein (pre-F), the protein structure was stabilized by introducing mutations at specific amino acid sites of the protein. While increasing the stability, the neutralization-sensitive epitopes of the pre-F conformation of the F protein were also retained. Summary of the invention

[0006] Based on this, the purpose of the present invention is to provide a mutant of RSV pre-fusion conformation F protein and its application, wherein the mutant of pre-fusion conformation F protein can improve the expression level and stability of pre-fusion trimerization conformation F protein.

[0007] To achieve the above objectives, the following technical solutions may be included.

[0008] The first aspect of the present invention is to provide a mutant of RSV pre-fusion conformation F protein, which comprises an amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein, wherein the amino acid mutations are substituted by cysteine ​​at positions 392 and 493 or the amino acids near position 493, respectively, and a disulfide bond is formed after the mutations between 392C and 493C or the mutations between 392C and 493C are mutated to cysteine.

[0009] In some of these embodiments, the position near position 493 includes position 491, which is mutated to 491C.

[0010] In some of these embodiments, the amino acid sequence of the wild-type RSV F protein is shown in SEQ ID NO: 1.

[0011] In some embodiments, the mutant further introduces a cavity filling mutation, and the cavity filling mutation is selected from at least one of the following: E60I, E60Y, S62V, K80M, K87M, K87Y, V90I, S150L, ​​S150V, S150F, S150W, V154L, K168Y, K168L, K191V, K191L, K191M, K427Y, D448V, S19 0F and V207L, E60I and K191V, S150L and K191V, K191V and D448V, D448Y, V154L and K191V, K168Y and K191V, K191V and K427Y, S150L and V154L, K427Y and D448V, E60I and S62V, S190F and V207L, or the above mutation sites are replaced by any hydrophobic amino acid.

[0012] Further preferably, the cavity filling mutation is selected from at least one of the following: K80M, K87M, K87Y, V90I, V154L, K427Y, D448Y, and 190F and V207L.

[0013] In some of the embodiments, the mutant includes a disulfide bond formed between the mutated D392C and S493C, and a cavity filling mutation, wherein the cavity filling mutation includes at least one selected from K80M, K87M, K87Y, V154L, D448Y, S190F and V207L.

[0014] In some of the embodiments, the mutants include a disulfide bond formed between the mutated D392C and S491C, and a cavity filling mutation, wherein the cavity filling mutation includes S190F and V207L ​​mutations.

[0015] In some of these embodiments, a proline mutation is also introduced, and the proline mutation is selected from at least one of the following: Q210P, S213P, I214P, S215P, N216P, I217P, N183P, S182P, N175P, S173P, G162P, E161P, S213P and I214P, I214P and S215P, S215P and N216P, N216P and I217P, E161P and G162P.

[0016] In some of the embodiments, the mutant includes a disulfide bond formed between the mutated D392C and S493C, and a cavity filling mutation of K87M, or S190F and V207L, and also includes at least one proline mutation selected from the following: Q210P, S213P, I214P, S215P, N216P, I217P, N183P, S182P, N175P, S173P, G162P, E161P, S213P and I214P, I214P and S215P, S215P and N216P, N216P and I217P, E161P and G162P.

[0017] In some of the embodiments, the mutants include the formation of a disulfide bond between the mutated D392C and S491C, as well as S190F, V207L ​​cavity filling mutations, and also include I214P mutation.

[0018] In some embodiments, the amino acid sequence of the mutant is shown in SEQ ID NO:3.

[0019] In some embodiments, the amino acid sequence of the mutant is shown in SEQ ID NO:5.

[0020] In some embodiments, the amino acid sequence of the mutant is shown in SEQ ID NO:7.

[0021] In some of the embodiments, the amino acid sequence of the mutant is shown in any one of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, and SEQ ID NO: 39.

[0022] The second aspect of the present invention is to provide a DNA sequence encoding a mutant of the RSV pre-fusion conformation F protein.

[0023] In some of these embodiments, the coding sequence is as shown in any one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:40.

[0024] The third aspect of the present invention is to provide a recombinant plasmid into which is inserted a coding sequence encoding any one of the mutants of the RSV pre-fusion conformation F protein.

[0025] The fourth aspect of the present invention is to provide an mRNA sequence encoding a mutant of the RSV pre-fusion conformation F protein, which can be obtained by enzyme digestion and transcription of a recombinant plasmid inserted with a coding sequence encoding any of the mutants.

[0026] The fifth aspect of the present invention is to provide the use of the above-mentioned mRNA sequence or the mutant in the preparation of RSV vaccine.

[0027] The sixth aspect of the present invention is to provide the use of the mutant in the detection of respiratory syncytial virus infection.

[0028] The sixth aspect of the present invention is to provide a respiratory syncytial virus (RSV) vaccine, characterized in that it comprises at least one ribonucleic acid polynucleotide having an open reading frame encoding any mutant of the above-mentioned RSV pre-fusion conformation F protein or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. By introducing the appropriate position (using amino acid 392 and amino acid 493 or 491) to achieve the effect of limiting the positional movement of the (HRB) region, the disulfide bond can stabilize the pre- and post-fusion conformation and trimerization conformation of the F protein, and significantly inhibit the conversion of the F protein to the post-fusion conformation.

[0031] 2. Based on the disulfide bond D392C-S491C, introducing a single-point cavity-filling mutation, or based on D392C-S493C, introducing specific single-point cavity-filling mutations and double-point cavity-filling mutations, will be more helpful in stabilizing the conformation of the F protein and increasing protein expression.

[0032] 3. The combination of disulfide bonds and cavity filling, electrostatic mutation, and introduction of proline mutations at specific positions described in the present invention can further significantly improve the expression and stability of F proteins having pre- and post-fusion conformations and trimerization conformations, and significantly inhibit the conversion of F proteins to post-fusion conformations. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :pNeoCura-Bvac plasmid map.

[0034] Figure 2 :Analysis of RF-NeoC-K1 mRNA transfected cells binding to monoclonal antibodies, including: A. Average fluorescence intensity of binding to monoclonal antibody D25, B. Average fluorescence intensity of binding to monoclonal antibody AM14, C. Average fluorescence intensity of binding to monoclonal antibody 4D7, D. Ratio of binding to monoclonal antibody D25, E. Average fluorescence intensity of binding to monoclonal antibody AM14, F. Ratio of binding to monoclonal antibody 4D7.

[0035] Figure 3 :Binding analysis of cells transfected with RF-NeoC-K24 and RF-NeoC-K35 mRNA and monoclonal antibodies, including A.D25 monoclonal antibody, B.AM14 monoclonal antibody, C.4D7 monoclonal antibody.

[0036] Figure 4 :Analysis of binding between mRF-NeoC-K133 mRNA transfected cells and monoclonal antibodies, including A.D25 monoclonal antibody, B.AM14 monoclonal antibody, C.4D7 monoclonal antibody.

[0037] Figure 5 : Protein expression level of mutant mRNA containing disulfide bond mutations relative to mRNA encoding RSV wild-type F protein, where A. protein bound to D25 monoclonal antibody, indicating the expression level of F protein in pre-fusion conformation; B. protein bound to AM14 monoclonal antibody, indicating the expression level of F protein in pre-fusion trimeric conformation; C. protein bound to 4D7 monoclonal antibody, indicating the expression level of F protein in post-fusion conformation.

[0038] Figure 6 : The protein expression levels of mutant mRNAs containing disulfide bond mutations and single cavity-filling mutations relative to the mRNA encoding RSV wild-type F protein, wherein A. the protein bound to the D25 monoclonal antibody indicates the expression level of the pre-fusion conformation F protein; B. the protein bound to the AM14 monoclonal antibody indicates the expression level of the pre-fusion trimer conformation F protein; C. the protein bound to the 4D7 monoclonal antibody indicates the expression level of the post-fusion conformation F protein.

[0039] Figure 7 : The protein expression level of mutant mRNA containing disulfide bond mutation and two cavity filling mutations relative to the mRNA encoding RSV wild-type F protein, wherein A. the protein bound to the D25 monoclonal antibody indicates the expression level of the pre-fusion conformation F protein; B. the protein bound to the AM14 monoclonal antibody indicates the expression level of the pre-fusion trimer conformation F protein; C. the protein bound to the 4D7 monoclonal antibody indicates the expression level of the post-fusion conformation F protein.

[0040] Figure 8 : The protein expression levels of mutant mRNAs containing disulfide bond mutations, single cavity-filling mutations, and proline mutations in the hinge region linking α-helices to α-helices or β-sheets relative to the mRNA encoding RSV wild-type F protein, wherein A. the protein bound to the D25 monoclonal antibody indicates the expression level of the F protein in the pre-fusion conformation; B. the protein bound to the AM14 monoclonal antibody indicates the expression level of the F protein in the trimeric conformation before fusion; C. the protein bound to the 4D7 monoclonal antibody indicates the expression level of the F protein in the post-fusion conformation. DETAILED DESCRIPTION

[0041] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0042] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0043] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0044] Definitions To facilitate understanding of this technology, certain terms and phrases are defined below.

[0045] A disulfide bond is a chemical bond that connects the sulfhydryl groups of two different cysteine ​​residues in different peptide chains or in the same peptide chain.

[0046] pre fusion pre-fusion conformation.

[0047] (1) Disulfide bond: As shown in Table 1, D392C and S493C are mutated to form a disulfide bond between them.

[0048] (2) Cavity filling: The two grooves of the conformation were modified (such as S190F and V207L ​​in Table 1) so that they were filled, as shown in RF-KW5 to RF-KW34.

[0049] The present invention introduces point mutations at amino acid sites into the F protein sequence of respiratory syncytial virus (RSV) to form disulfide bonds, and / or cavity filling, and / or a combination of disulfide bonds and cavity filling, thereby significantly improving the expression of pre-fusion conformation F protein; the pre-fusion F protein can form a stable trimer and has a stable trimerization conformation. Using the mutant F protein as an immunogen or vaccine can significantly improve the level of induced neutralizing antibodies against RSV.

[0050] The embodiment of the present invention uses the F protein sequence of the RSV A2 strain as the basic sequence for the mutant mutation, but the basic sequence for the mutant mutation of the present invention is not limited to the RSV A2 strain, but also includes other types of RSV such as type B and clinical strains.

[0051] As is well known, RSV has two antigenic subtypes, A and B. The amino acid sequences of the mature processed F proteins of the two subtypes are about 93% identical. As used in the present invention, the amino acid position is given with reference to the sequence (SEQ ID NO: 1) of the RSV F protein (F protein or mutant of F protein) from the A2 strain. As used in the present invention, the wording "amino acid at position "x" of the RSV F protein thus means the amino acid corresponding to the amino acid at position "x" in the RSV F protein of the RSVA2 strain of SEQ ID NO: 1. Note that in the sequence numbering system used in the present invention, 1 refers to the N-terminal amino acid of an immature F protein (corresponding to the first amino acid of SEQ ID NO: 1). When a RSV strain is used instead of the A2 strain, the amino acid position of the F protein will be numbered by inserting spaces where necessary to align the sequences of other RSV strains with the F protein of SEQ ID NO: 1, with reference to the numbering of the F protein of the A2 strain of SEQ ID NO: 1. Sequence alignment can be performed using methods well known in the art, such as by CLUSTALW, Bioedit or CLC workbench.

[0052] The following lists the amino acid sequence and nucleic acid sequence of some of the F proteins after mutation and modification. For other amino acid and nucleic acid sequences not given, the corresponding amino acid and nucleic acid sequences can be obtained according to the mutation positions given in Table 1, with reference to the given wild-type sequence and the following sequence, according to conventional techniques.

[0053] SEQ ID NO.1

[0054] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTVTTLSKKRKRRFLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYS IMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0055] SEQ ID NO.2

[0056]

[0057] SEQ ID NO.3(RF-NeoC-K1)

[0058] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKY C CKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASI C QVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0059] SEQ ID NO.4(dRF-NeoC-K1)

[0060]

[0061] SEQ ID NO.5(RF-NeoC-K24)

[0062] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLT F KVLDLKNYIDKQLLPI L NKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKY C CKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASI C QVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0063] SEQ ID NO.6(dRF-NeoC-K24)

[0064]

[0065] SEQ ID NO:7(RF-NeoC-K35)

[0066] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLT F KVLDLKNYIDKQLLPI L NKQSCS P SNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKY C CKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASI C QVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0067] SEQ ID NO:8(dRF-NeoC-K35)

[0068]

[0069] SEQ ID NO:9(RF-NeoC-K131)

[0070] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDACISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0071] SEQ ID NO:10(dRF-NeoC-K131)

[0072]

[0073] SEQ ID NO:11(RF-NeoC-K132)

[0074] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDACISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0075] SEQ ID NO:12(dRF-NeoC-K132)

[0076]

[0077] SEQ ID NO:13(RF-NeoC-K133)

[0078] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSPSNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDACISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0079] SEQ ID NO:14(dRF-NeoC-K133)

[0080]

[0081] SEQ ID NO:15(RF-NeoC-K5)

[0082] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIMQELDKYKNAVTELQLLMQSTQATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0083] SEQ ID NO:16(dRF-NeoC-K5)

[0084]

[0085] SEQ ID NO:17(RF-NeoC-K6)

[0086] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYMNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0087] SEQ ID NO:18(dRF-NeoC-K6)

[0088]

[0089] SEQ ID NO:19(RF-NeoC-K7)

[0090] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYYNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0091] SEQ ID NO:20(dRF-NeoC-K7)

[0092]

[0093] SEQ ID NO:21(RF-NeoC-K21)

[0094] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVYTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0095] SEQ ID NO:22(dRF-NeoC-K21)

[0096]

[0097] SEQ ID NO:23(RF-NeoC-K82)

[0098] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYMNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSPSNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0099] SEQ ID NO:24(dRF-NeoC-K82)

[0100]

[0101] SEQ ID NO:25(RF-NeoC-K83)

[0102] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYMNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0103] SEQ ID NO:26(dRF-NeoC-K83)

[0104]

[0105] SEQ ID NO:27(RF-NeoC-K84)

[0106] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYMNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISPIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0107] SEQ ID NO:28(dRF-NeoC-K84)

[0108]

[0109] SEQ ID NO:29(RF-NeoC-K85)

[0110] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYMNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNPETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0111] SEQ ID NO:30(dRF-NeoC-K85)

[0112]

[0113] SEQ ID NO:31(RF-NeoC-K89)

[0114] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYMNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTPKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0115] SEQ ID NO:32(dRF-NeoC-K89)

[0116]

[0117] SEQ ID NO:33(RF-NeoC-K93)

[0118] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYMNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLPGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0119] SEQ ID NO:34(dRF-NeoC-K93)

[0120]

[0121] SEQ ID NO:35(RF-NeoC-K94)

[0122] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYMNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKPSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0123] SEQ ID NO:36(dRF-NeoC-K94)

[0124]

[0125] SEQ ID NO:37(RF-NeoC-K96)

[0126] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYMNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSPPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0127] SEQ ID NO:38(dRF-NeoC-K96)

[0128]

[0129] SEQ ID NO:39(RF-NeoC-K103)

[0130] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKKNKCNGTDAKVKLIKQELDKYMNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLPPEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYCCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASICQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0131] SEQ ID NO:40(dRF-NeoC-K103)

[0132]

[0133] The present invention is further described in detail below with reference to specific embodiments.

[0134] Example 1 Preparation of plasmid templates encoding RSV wild-type F protein and mutants.

[0135] Based on the wild-type F protein sequence of the RSV A2 strain (as shown in SEQ ID NO: 1) and the optimized DNA sequence encoding the wild-type F protein sequence of the RSV A2 strain (as shown in SEQ ID NO: 2), the DNA sequence of the wild-type F protein sequence was recombined between the restriction sites BamHⅠ and SacⅠ sequences of the pNeoCura-Bvac plasmid vector by gene synthesis to obtain the recombinant plasmid pNeoCura-Bvac-WT, which was used as a plasmid template for preparing mRNA (mWT) encoding the wild-type F protein.

[0136] According to the corresponding sites of the amino acid sequences of the mutants shown in Table 1, point mutations were performed on the recombinant plasmid pNeoCura-Bvac-WT to obtain the corresponding recombinant plasmids pNeoCura-Bvac-RF-NeoC-K1 to pNeoCura-Bvac-RF-NeoC-K133 (DNA sequences: dRF-NeoC-K1 to dRF-NeoC-K133). We also constructed the recombinant plasmid pNeoCura-Bvac-Ds-Cav1, which encodes the same amino acid mutation sites as Ds-Cav1, but the C-terminus of F1 retains the original transmembrane region (TM region) and intracellular region (cytoplasmic region CT region) amino acid sequences of F1.

[0137] Table 1:

[0138]

[0139]

[0140]

[0141] Example 2 Preparation of mRNA encoding RSV wild-type F protein and mutants

[0142] 2.1. Linearized template preparation

[0143] The recombinant plasmids were digested with restriction endonuclease BspQ I (NEB, catalog number: R0712L), and the linearized plasmids were purified using VAHTS DNA Clean Beads (Novagen, N411-01).

[0144] 2.2 Preparation of mRNA by in vitro transcription

[0145] According to the instructions of the in vitro transcription kit T7 High Yield RNA Transcription Kit (N1-Me-PseudoUTP) (Novozyme, DD4202), the linearized plasmid prepared in 2.1 was used as a template to perform in vitro transcription to obtain mRNA. VAHTS RNA Clean Beads (Vazyme, N412-01) were used to purify mRNA according to the instructions.

[0146] 2.3 Capping and purification

[0147] The capping kit mRNA Cap2'-O-Methyltransferase (Novozyme, DD4110) was used to perform capping reaction on the purified mRNA. The capped mRNA was purified using VAHTS RNA Clean Beads (Vazyme, catalog number: N412-01) to obtain mRNA of the corresponding sequence.

[0148] Example 3 mRNA transfected cells and detection of cell surface F protein expression

[0149] 3.1 HEK293T cell plating

[0150] HEK293T cells were seeded in 24-well plates (2.5-3×10 5 / well), and cultured in DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C, 5% CO2 for 18 hours. The cell density reached 80% confluence before transfection.

[0151] 3.2 mRNA transfection into HEK293T cells

[0152] The cell culture medium was replaced with serum-free Opti-MEM medium (Thermo Fisher Scientific), and mRNA (0.3 μg or 1 μg RNA / well) and Lipofectamine TM Messenger MAX TM The cells were transfected with transfection reagent (Thermo Fisher Scientific) and then mixed. After 6 hours, the cell culture medium was replaced with DMEM medium (Thermo Fisher Scientific) containing 10% fetal bovine serum and 1% penicillin-streptomycin, and the culture was continued for 24 hours.

[0153] 3.3 Detection of F protein expression on the surface of HEK293T cells by flow cytometry

[0154] After culturing the transfected cells for 24 hours, the cells were centrifuged at 1000 g for 10 minutes, and the cells were collected and incubated with fluorescently labeled monoclonal antibodies. Flow cytometry (BD, FACS Celesta) was used to detect the mean fluorescence intensity (MFI) and cell ratio of F protein on the cell surface bound to monoclonal antibody D25, monoclonal antibody AM14 or monoclonal antibody 4D7. The monoclonal antibody D25 in the present invention: (D25 was prepared using the method disclosed in McLellan, JS, Chen, M., Leung, S., Graepel, KW, Du, X., Yang, Y., Zhou, T., Baxa, U., Yasuda, E., Beaumont, T., Kumar, A., Modjarrad, K., Zheng, Z., Zhao, M., Xia, N., Kwong, PD, & Graham, BS (2013). Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science (New York, NY), 340(6136), 1113-1117. https: / / doi.org / 10.1126 / science.1234914), the mean fluorescence intensity value (MFI) of the binding of D25 to the cell surface F protein characterizes the expression level of the F protein in the prefusion conformational epitope;

[0155] The monoclonal antibody AM14 in the present invention: (AM14 was prepared using the method disclosed in Gilman, MS, Moin, SM, Mas, V., Chen, M., Patel, NK, Kramer, K., Zhu, Q., Kabeche, SC, Kumar, A., Palomo, C., Beaumont, T., Baxa, U., Ulbrandt, ND, Melero, JA, Graham, BS, & McLellan, JS (2015). Characterization of a Prefusion-Specific Antibody That Recognizes aQuaternary, Cleavage-Dependent Epitope on the RSV Fusion Glycoprotein. PLoSpathogens, 11(7), e1005035. https: / / doi.org / 10.1371 / journal.ppat.1005035). The mean fluorescence intensity value (MFI) of AM14 binding to cell surface F protein characterizes the expression level of F protein in the pre-fusion trimerization conformation epitope;

[0156] The monoclonal antibody 4D7 in the present invention: (4D7 was prepared using the method disclosed in Zhang, L., Durr, E., Galli, JD, Cosmi, S., Cejas, PJ, Luo, B., Touch, S., Parmet, P., Fridman, A., Espeseth, AS, & Bett, AJ (2018). Design and characterization of a fusion glycoprotein vaccine for Respiratory Syncytial Virus with improved stability. Vaccine, 36(52), 8119-8130. https: / / doi.org / 10.1016 / j.vaccine.2018.10.032). The mean fluorescence intensity value (MFI) of the binding of 4D7 to the cell surface F protein characterizes the expression level of the F protein in the post-fusion conformational epitope.

[0157] Experimental results:

[0158] (1) The second heptad repeat (HRB) region is one of the unstable regions, which forms the trimerized helical stem region in the pre-fusion conformation of the F protein. In the transition from the pre-fusion conformation to the post-fusion conformation, the second heptad repeat (HRB) region participates in the formation of three α helices in the six-helix bundle in the post-fusion conformation. The amino acid at position 392 forms a disulfide bond with the amino acid at position 493 or 491 to limit the positional movement of the (HRB) region, thereby stabilizing the pre-fusion conformation.

[0159] like Figure 2 The results showed that compared with WT mRNA encoding wild-type F protein, the mean fluorescence intensity (MFI) and ratio of D25 binding in cells transfected with RF-NeoC-K1 mRNA were significantly increased ( Figure 2 A and Figure 2 D), indicating that the disulfide bond D392C-S493C mutation significantly increased the expression level and proportion of the pre-fusion conformation protein of the F protein. The mean fluorescence intensity (MFI) and proportion of RF-NeoC-K1mRNA transfected cells binding to AM14 were significantly increased ( Figure 2 B and Figure 2 E), indicating that the disulfide bond D392C-S493C mutation can also significantly increase the expression level and ratio of the pre-fusion conformation protein trimer of the F protein. In addition, due to the unstable conformation of the wild-type F protein encoded by WT mRNA, the mean fluorescence intensity (MFI) and ratio of WT mRNA-transfected cells binding to 4D7 are at a high level, while the mean fluorescence intensity (MFI) and ratio of mRF-NeoC-K1 mRNA-transfected cells binding to 4D7 are significantly reduced ( Figure 2 C and Figure 2 F), which is close to the untransfected mRNA group (nomRNA). This indicates that the disulfide bond D392C-S493C mutation significantly reduces the expression level and proportion of the post-fusion conformation protein trimer of the F protein.

[0160] The combination of disulfide bond D392C-S493C and cavity filling and proline mutation at specific positions, such as the mean fluorescence intensity (MFI) of cells transfected with RF-NeoC-K24 and RF-NeoC-K35 binding to D25 and AM14, was significantly higher than that of Ds-Cav1 in the prior art (a stabilized pre-fusion F protein containing the RSV F transmembrane domain that has been disclosed), indicating that the level of pre-fusion conformation trimerized F protein encoded by RF-NeoC-K24 and RF-NeoC-K35 was significantly higher than that of Ds-Cav1 ( Figure 3A and 3B); the mean fluorescence intensity (MFI) of cells transfected with RF-NeoC-K24 and RF-NeoC-K35 and the antibody 4D7 characterizing the post-fusion conformation was significantly lower than that of Ds-Cav1, indicating that the level of trimerized F protein encoded by RF-NeoC-K24 and RF-NeoC-K35 after fusion was significantly lower than that of Ds-Cav1, and had a more stable pre-fusion conformation ( Figure 3 C).

[0161] We also found that when the amino acid near position 493 (such as position 491) mutates to cysteine, a disulfide bond is formed between 392C and 491C, which can also help stabilize the pre-fusion conformation of the F protein. Figure 4 Compared with the wild-type F protein sequence (WT), the combination of disulfide bond D392C-S491C and cavity filling and proline mutations at specific positions, such as the mean fluorescence intensity (MFI) of cells transfected with RF-NeoC-K133 binding to D25 and AM14, was significantly increased, indicating that the level of pre-fusion conformation trimerized F protein encoded by RF-NeoC-K133 was significantly higher than that of mDs-Cav1 ( Figure 4 Middle A, Figure 4 Middle B); The mean fluorescence intensity (MFI) of cells transfected with RF-NeoC-K131, RF-NeoC-K132, and RF-NeoC-K133 and the antibody 4D7 that characterizes the post-fusion conformation was significantly reduced, indicating that the post-fusion conformation level of the F protein encoded by RF-NeoC-K131, RF-NeoC-K132, and RF-NeoC-K133 was significantly reduced, and had a more stable pre-fusion conformation ( Figure 4 C). Although only the 392C and 491C mutations did not significantly increase the mean fluorescence intensity (MFI) of the cell surface F protein binding to D25 and AM14, it may be because the overall level of F protein did not increase after RF-NeoC-K131 transfected cells; however, the 392C and 491C mutations significantly reduced the expression of the post-fusion F protein conformation, indicating that the 392C and 491C mutations can effectively stabilize the F protein in the pre-fusion trimerization conformation. In summary, the mutation of amino acid sites near positions 392C and 493 (such as position 491) to cysteine ​​(C) can also effectively stabilize the pre-fusion conformation of the F protein and effectively inhibit the transformation of the post-fusion conformation of the F protein.

[0162] To further evaluate the role of cysteine ​​substitution of amino acids at positions 392 and 493 or amino acids near position 493, respectively, between 392C and 493C after mutation, or between 392C and positions near position 493 after mutation, to form a disulfide bond, cysteine ​​mutation D392C-S491C was introduced using amino acids at positions 392 and 491.

[0163] The results showed that compared with WT mRNA encoding wild-type F protein, the mean fluorescence intensity (MFI) and ratio of RF-NeoC-K131 mRNA transfected cells to D25, AM14, and 4D7 were extremely low ( Figure 5 ); indicating that the D392C-S491C mutation significantly reduced the expression of the post-fusion conformation F protein.

[0164] Introducing hydrophobic amino acid mutations to fill the cavity helps stabilize the conformation of the F protein and increase protein expression. Based on the disulfide bond D392C-S491C, a single-point cavity-filling mutation was introduced, such as Figure 6 The results showed that the cavity-filling mutations K80M, K87M, K87Y, V154L, and K427Y could further increase the prefusion of F protein ( Figure 6 A) Trimeric conformation before fusion ( Figure 6 B) Expression of F protein, while reducing the post-fusion conformation ( Figure 6 C) Expression of F protein.

[0165] Further, based on the disulfide bond D392C-S491C, two cavity-filling mutations were introduced, such as Figure 7 The results showed that the combination of cavity filling mutations S190F and V207 could further increase the prefusion of F protein ( Figure 7 A) Trimeric conformation before fusion ( Figure 7 B) Expression of F protein, while reducing the post-fusion conformation ( Figure 7 C) Expression of F protein.

[0166] On the basis of the disulfide bond D392C-S491C, two cavity-filling mutations were introduced: E60I, K191V; E60I, K191V; S150L, ​​K191V, K191V, D448V; V154L, K191V; K168Y, K191V; K191V, K427Y; S150L, ​​V154L; K427Y, D448V; E60I, S62V reduced the protein pre-fusion ( Figure 7 A) Trimeric conformation before fusion ( Figure 7 B) post-fusion conformation ( Figure 7 C) Expression of F protein.

[0167] Furthermore, the introduction of specific proline mutations at the hinge region linking the α-helix and α-helix or β-fold helps stabilize the conformation of the F protein and increase protein expression. Proline mutations were added on the basis of the disulfide bond D392C-S491C and cavity filling K87M mutations (RF-NeoC-K6). The results showed that compared with the design RF-NeoC-K6 without the addition of proline mutation sites, RF-NeoC-K82 (containing I214P), RF-NeoC-K83 (containing S215P), RF-NeoC-K93 (containing E161P), and RF-NeoC-K94 (containing Q210P) all further increased the expression of the pre-fusion conformation F protein; RF-NeoC-K82 (containing I214P), RF-NeoC -K83 (containing S215P), RF-NeoC-K85 (containing I217P), RF-NeoC-K93 (containing E161P), RF-NeoC-K95 (containing S213P, I214P), RF-NeoC-K96 (containing I214P, S215P), RF-NeoC-K97 (containing S21PP, N216P) all further increased the expression of the pre-fusion trimer conformation F protein; at the same time, as Figure 8 As shown, the addition of the proline mutation site did not increase the expression of the post-fusion conformation F protein.

[0168] Based on the design of RF-NeoC-K132 containing disulfide bonds D392C-S491C and cavity-filling mutations S190F and V207, the proline mutation I214P was added to improve the prefusion of the F protein ( Figure 8 A) Trimeric conformation before fusion ( Figure 8 B) Expression of F protein, while reducing the post-fusion conformation ( Figure 8 C) Expression of F protein.

[0169] In summary, the disulfide bonds designed by the present invention, and / or the combination with cavity filling and proline mutation at specific positions can significantly improve the expression level of F protein with a pre-fusion trimerization conformation and maintain the stability of the trimer conformation of the F protein before fusion.

[0170] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A mutant of RSV prefusion conformation F protein, characterized in that: The mutant comprises an amino acid mutation relative to the amino acid sequence of the wild-type RSV F protein, wherein the amino acids at positions 392 and 493 are mutated to amino acids at positions 392 and 493, or the amino acids at positions near position 493 are replaced by cysteine, respectively, and a disulfide bond is formed after the positions between the mutated 392C and 493C, or the positions near the mutated 392C and 493 are mutated to cysteine.

2. The mutant according to claim 1, characterized in that: The positions near position 493 include position 491, which is mutated to 491C.

3. The mutant according to claim 1, characterized in that: The mutant also introduces a cavity filling mutation, and the cavity filling mutation is selected from at least one of the following: E60I, E60Y, S62V, K80M, K87M, K87Y, V90I, S150L, ​​S150V, S150F, S150W, V154L, K168Y, K168L, K191V, K191L, K191M, K427Y, D448V, D448Y, S190F and V207L, E60I and K191V, S150L and K191V, K1 91V and D448V, V154L and K191V, K168Y and K191V, K191V and K427Y, S150L and V154L, K427Y and D448V, E60I and S62V, S190F and V207L, or the above mutation sites are replaced by any hydrophobic amino acid; preferably, the cavity filling mutation is selected from at least one of the following: K80M, K87M, K87Y, V90I, V154L, K427Y, D448Y and 190F and V207L.

4. The mutant according to claim 1, characterized in that: The mutant includes a disulfide bond formed between the mutated D392C and S493C, and a cavity filling mutation, wherein the cavity filling mutation includes at least one selected from K80M, K87M, K87Y, V154L, D448Y, S190F and V207L; or The mutants include a disulfide bond formed between the mutated D392C and S491C, and a cavity filling mutation, wherein the cavity filling mutation includes S190F and V207L ​​mutations.

5. The mutant according to any one of claims 1 to 4, characterized in that: The mutant also introduces a proline mutation, and the proline mutation is selected from at least one of the following: Q210P, S213P, I214P, S215P, N216P, I217P, N183P, S182P, N175P, S173P, G162P, E161P, S213P and I214P, I214P and S215P, S215P and N216P, N216P and I217P, E161P and G162P.

6. The mutant according to any one of claims 1 to 4, characterized in that: The mutant includes a disulfide bond formed between the mutated D392C and S493C, and a cavity-filling mutation of K87M, or S190F and V207L, and also includes at least one proline mutation selected from the following: Q210P, S213P, I214P, S215P, N216P, I217P, N183P, S182P, N175P, S173P, G162P, E161P, S213P and I214P, I214P and S215P, S215P and N216P, N216P and I217P, E161P and G162P; or The mutants include a disulfide bond formed between the mutated D392C and S491C, as well as S190F and V207L ​​cavity filling mutations, and also include I214P mutation.

7. The mutant according to claim 1, characterized in that: The amino acid sequence of the mutant is shown in any one of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, and SEQ ID NO:

39.

8. A DNA coding sequence encoding a mutant of the RSV prefusion conformation F protein according to any one of claims 1 to 7.

9. The DNA coding sequence according to claim 8, characterized in that The coding sequence is shown in any one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:

40.

10. A recombinant plasmid, into which the DNA coding sequence according to any one of claims 8 to 9 is inserted.

11. An mRNA sequence encoding a mutant of the RSV prefusion conformation F protein according to any one of claims 1 to 7.

12. A respiratory syncytial virus (RSV) vaccine, characterized in that It comprises at least one ribonucleic acid polynucleotide having an open reading frame encoding a mutant of the RSV F protein pre-fusion conformation or an immunogenic fragment thereof according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

13. Use of the mutant of RSV prefusion conformation F protein according to any one of claims 1 to 7 and the mRNA sequence according to claim 10 in the preparation of RSV vaccine.

14. Use of the mutant of RSV prefusion conformation F protein according to any one of claims 1 to 7 in detecting respiratory syncytial virus infection.

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