Mutant of RSV pre-fusion f protein, and preparation and use thereof

Abstract

The present invention relates to a mutant of an RSV pre-fusion F protein, and the preparation and the use thereof. Provided is the mutant of the RSV pre-fusion F protein. An F1 polypeptide thereof has cysteine substituent 1 in an α3 helix region, and cysteine substituent 2 in a β3 sheet region. The cysteine substituent 1 and the cysteine substituent 2 form a disulfide bond.

Description

Mutants of rsv pre-fusion f protein and preparation and use thereof Related applications This application claims priority to the Chinese patent application No. 202410642386X, filed on May 22, 2024, entitled “Mutants of rsv pre-fusion f protein and preparation and use thereof”, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD The present application belongs to the field of biotechnology, and relates to a mutant of RSV pre-fusion F protein and preparation and use thereof. BACKGROUND Respiratory syncytial virus (RSV) is a highly contagious respiratory pathogen and is one of the main causes of hospitalization and death in infants under 6 months worldwide, which can cause severe lower respiratory tract infection in infants, the elderly and immunodeficient patients. WHO study on the etiology of acute respiratory infections in children showed that the number of cases of acute respiratory infection caused by RSV accounted for more than 60% of the total number of respiratory diseases in children. In 2019, there were 33 million cases of acute lower respiratory tract infection (ALRI) caused by RSV infection in children aged 0-5 worldwide, 3.6 million children were hospitalized, and a total of 101,000 children died in the hospital, of which about 46% were infants under 6 months of age, which caused a huge economic burden on public health resources in each country. RSV belongs to the Paramyxoviridae family, and the virus genome consists of 15,200 nucleotides, including 10 genes encoding 11 proteins. Among them, the structural proteins include three transmembrane surface glycoproteins: attachment protein G (Glycoprotein, G), fusion protein F (Fusion protein, F) and small hydrophobic protein SH (small hydrophobic protein, SH). Fusion protein F and attachment protein G, two transmembrane surface glycoproteins, are key targets for neutralizing antibodies. However, the highly variable glycosylation of attachment protein G and fusion protein F conformational changes pose challenges to vaccine development. The fusion protein F is a type I membrane fusion protein, the F0 protein encoded by the virus forms a trimer in the endoplasmic reticulum, and contains two Furin protease cleavage sites, which can form F1, F2 two proteins and a Pep27 polypeptide fragment after Furin protease cleavage, the F1 protein contains its N-terminal hydrophobic fusion peptide and two heptad repeat regions (HRA and HRB). Under physiological conditions, the pre-F protein (pre-Fusion conformation, pre-F) is in a metastable state and is easily converted into a stable post-fusion conformation (Postfusion conformation, post-F), which loses the main neutralizing antigen epitope However, in the serum of patients naturally infected with RSV, most neutralizing antibodies are directed against the pre-fusion conformation. At present, the crystal structures of the two conformations of the F protein have been resolved, and based on the molecular structure, several pre-F conformationally stable mutants have been obtained through point mutations. For example: the DS-Cav1 mutant (S155C, S290C, S190F, V207L) described in CN105473604B, US10017543B2, US11130785B2; the pre-F stable mutant (N67I, S215P, E487Q) containing SC-DM and SC-TM described in US20150320854A1, WO2014174018A1, AU2014259474B2, CN105408348B; the pre-F mutant (S55C, L188C, L142C, N371C) described in US20210023200A1, CN108738312A; the pre-F mutant containing L141C, L142C and L373C position mutations described in CN114929877A. Traditional mutation strategies mainly include disulfide bond mutation, cavity filling or charge balance, the purpose of which is to maintain the stabilized conformation of pre-F or to improve the expression level of pre-F. However, the stabilization of pre-F protein is still insufficient, and structure-based optimization is still under continuous research. In view of this, the present application is proposed. SUMMARY The main purpose of the embodiments of the present application is a mutant of RSV pre-fusion F protein and its preparation and application. The technical solution comprises: In the first aspect of the embodiments of the present application, a mutant of RSV pre-fusion F protein is provided, wherein the alpha 3 helix region of the F1 polypeptide has a cysteine substituent 1, and the beta 3 sheet region has a cysteine substituent 2, and the cysteine substituent 1 and the cysteine substituent 2 form a disulfide bond. In some embodiments of the application, the species origin of the RSV pre-fusion F protein is human or bovine. In some embodiments of the application, the F1 polypeptide of the RSV pre-fusion F protein has an amino acid sequence as set forth in SEQ ID NO. 51 or has at least 80% homology to the amino acid sequence as set forth in SEQ ID NO. 51. In some embodiments of the application, the F1 polypeptide of the RSV pre-fusion F protein has an amino acid sequence as set forth in SEQ ID NO. 51, SEQ ID NO. 74, or SEQ ID NO. 75. In some embodiments of the application, the F1 polypeptide of the RSV pre-fusion F protein has an amino acid sequence as set forth in SEQ ID NO. 51, SEQ ID NO. 74, or SEQ ID NO. 75 and has one or more mutations selected from the group consisting of: In some embodiments of the application, the F1 polypeptide of the RSV pre-fusion F protein has one or more mutations selected from the group consisting of: (1) does not contain a transmembrane domain, and (2) does not contain an intracellular domain. In some embodiments of the application, the F1 polypeptide of the RSV pre-fusion F protein has one or more mutations selected from the group consisting of I379V and M447V. In some embodiments of the application, the F1 polypeptide of the RSV pre-fusion F protein has an amino acid sequence as set forth in SEQ ID NO. 52. In some embodiments of the application, the distance between the cysteine substituent 1 and the cysteine substituent 2 is In some embodiments of the application, the a3 helix region of the F1 polypeptide of the mutant has one or more mutations selected from the group consisting of E163C, K166C, I167C, A170C, and L171C. In some embodiments of the application, the b3 sheet region of the F1 polypeptide of the mutant has one or more mutations selected from the group consisting of A177C, V179C, and L181C. In some embodiments of the application, the F1 polypeptide of the mutant has one set of mutations selected from the group consisting of: Set 1 is E163C and L181C, Set 2 is K166C and V179C, Set 3 is I167C and V179C, Set 4 is A170C and V179C, Set 5 is A170C and A177C, and Set 6 is L171C and A177C. In some embodiments of the application, the mutant further has one or more of the following mutation sites: S55C, S155C, L188C, S190F, V207L, and S290C. In some embodiments of the application, the mutant has one set of the following combinations of mutation sites: Group 1) S155C and S290C, Group 2) S55C and L188C, and Group 3) S190F and V207L. In some embodiments of the application, the mutant does not contain the furin cleavage site fragment. In some embodiments of the application, the mutant does not contain the pep27 polypeptide, and the C-terminus of the F2 polypeptide and the N-terminus of the Fl polypeptide are directly connected by an amide bond or indirectly connected by a flexible short peptide. In some embodiments of the application, the flexible short peptide is optionally GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG, or GGGGS. In some embodiments of the application, the C-terminus of the Fl polypeptide of the mutant is connected to a tag fragment and / or an aggregation motif. In some embodiments of the application, the tag fragment comprises a 6His polypeptide. In some embodiments of the application, the aggregation motif is as shown in SEQ ID NO. 48. In some embodiments of the application, the F2 polypeptide of the mutant has an amino acid sequence as shown in SEQ ID NO. 49 or has at least 80% homology with the amino acid sequence as shown in SEQ ID NO. 49. In some embodiments of the application, the F2 polypeptide of the mutant has an amino acid sequence as shown in SEQ ID NO. 49, SEQ ID NO. 76, or SEQ ID NO. 77. In some embodiments of the application, the F2 polypeptide of the mutant satisfies one or more of the following conditions: 1) does not contain NN at the C-terminus, and 2) has the following mutation: P102A. In some embodiments of the application, the F2 polypeptide of the mutant has an amino acid sequence as shown in SEQ ID NO. 50. In some embodiments of the present application, the amino acid sequence of the mutant is as shown in any one of SEQ ID NO. 12 to SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42 to SEQ ID NO. 47, and SEQ ID NO. 53 to SEQ ID NO. 73. In a second aspect of the embodiments of the present application, a nucleic acid molecule encoding the mutant of the RSV pre-fusion F protein of the first aspect is provided. variant. In a third aspect of the embodiments of the present application, a vector comprising the nucleic acid molecule of the second aspect is provided. In a fourth aspect of the embodiments of the present application, an engineered cell expressing the mutant of the RSV pre-fusion F protein of the first aspect, or comprising the nucleic acid molecule of the second aspect, or the vector of the third aspect is provided. In a fifth aspect of the embodiments of the present application, a method for producing the mutant of the RSV pre-fusion F protein is provided, comprising the steps of: culturing the engineered cell of the fourth aspect, and isolating the mutant of the RSV pre-fusion F protein from the supernatant of the resulting culture. In a sixth aspect of the embodiments of the present application, an immunological composition comprising the mutant of the RSV pre-fusion F protein of the first aspect or the nucleic acid molecule of the second aspect, and an immunological adjuvant is provided. In some embodiments of the present application, the immunological adjuvant comprises one or more of an aluminum salt adjuvant, a surfactant, a polynucleotide, a lipopolysaccharide, a liposome, and an oil emulsion adjuvant. In a seventh aspect of the embodiments of the present application, the mutant of the RSV pre-fusion F protein of the first aspect is used in the preparation of a respiratory syncytial virus antibody detection kit. In an eighth aspect of the embodiments of the present application, a respiratory syncytial virus antibody detection kit comprising the mutant of the RSV pre-fusion F protein of the first aspect is provided. In a ninth aspect of the embodiments of the present application, a method for preventing or treating lower respiratory tract infection caused by respiratory syncytial virus is provided, comprising the step of: administering to a subject a therapeutically effective amount of the immunological composition of the sixth aspect. In a tenth aspect of the embodiments of the present application, a method for detecting or isolating RSV F binding antibody in a sample is provided, comprising the steps of: contacting the mutant of the RSV pre-fusion F protein of the first aspect with the RSV F binding antibody in the sample to form an immunocomplex; and, The immune complex is detected to detect or isolate RSV F-binding antibodies in a sample. Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 is a schematic diagram of the RSV F0 protein structure (A, B, bovine); Figure 2 is a schematic diagram of the structure of the constructed stable mutant RSV F protein polypeptide monomer; Figure 3 is a schematic diagram of the structure of the stabilized mutant RSV F protein polypeptide trimer constructed; Figure 4 is a diagram showing the presence of 7 pairs of disulfide bonds in the natural RSV F0 precursor protein or mature F protein. Figure 5 shows a schematic diagram of the three-dimensional structure of the RSV F protein monomer (left) and a magnified schematic diagram of the local area located in the α3 and β3 sheets (right); Figure 6 shows the amino acids involved in the α3 helix and β3 fold positions in this application, as well as the interatomic distances between two amino acids that readily form disulfide bonds. Figure 7 shows the SDS-PAGE analysis results of the mutant under reducing conditions; where JW-05 is the mutant monomer and JW-05-T4 is the mutant trimer. Figure 8 shows the high-performance liquid chromatography (HPLC) analysis results of the RSV pre-F mutant; Figure 8a shows the mutant monomer, and Figure 8b shows the mutant trimer. Figure 9 shows the expression statistics of RSV pre-F monomers formed by the α3 / β3 position mutation; Figure 10 shows the thermal stability of RSV pre-F monomers formed by the α3 / β3 positional mutation under 50℃ treatment. Figure 11 shows the statistical graph of RSV pre-F mutant trimer expression levels; Figure 12 shows the thermal stability of RSV pre-F mutant trimer under 50℃ treatment; Figure 13 shows the RSV pre-F mutant trimer and D25 monoclonal antibody ( The binding specificity of epitopes; Figure 14 is the binding specificity of RSV pre-F mutant trimers to AM22 monoclonal antibody (epitope); epitope); Figure 15 is the binding specificity of RSV pre-F mutant trimers to AM14 monoclonal antibody (trimer epitope); Figure 16 is the results of RSV pre-F mutant antigen-specific antibody level detection; Figure 17 is the results of RSV pre-F mutant neutralizing antibody detection; Figure 18 is the adsorption rate of RSV pre-F mutant to neutralizing antibodies in serum. DETAILED DESCRIPTION The present application will be further described in conjunction with the drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to explain the present application and not intended to limit the scope of the present application, the purpose of providing these embodiments and examples is to make the understanding of the present disclosure more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein, those skilled in the art can make various modifications or changes without departing from the spirit of the present application, and the equivalent forms obtained thereby also fall within the scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, it should be understood that the present application can be implemented without one or more of these details. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing embodiments and examples only and is not intended to be limiting of the application. Terminology Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings: The selection range of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, including any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and"). In the present application, "multiple", "various", "multiple times", "multiple", etc. are used without specific limitation, which means more than two or equal to two in quantity. For example, "one or more" means one or more than two. As used herein, "combinations thereof", "any combination thereof", "any combination thereof", etc. include all suitable combinations of any two or more listed items. In the present application, "suitable", "suitable", "any suitable way", etc. are described as being able to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the expected technical effects of the present application. In the present application, "preferably", "better", "better", "preferably" are only used to describe the implementation mode or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of the present application. In the present application, "further", "further", "particularly", etc. are used to describe the purpose, indicating the difference in content, but should not be understood as a limitation on the protection scope of the present application. In the present application, "optionally", "optional", "optional" means optional, i.e. selected from two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified, and there is no contradiction or mutual restriction, each "optional" is independent. In the present application, the terms "first", "second", "third", "fourth" and the like in the "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and should be understood as not constituting a closed limitation on the quantity. In the present application, among the technical features described in an open manner, both the closed technical solution consisting of the listed features and the open technical solution containing the listed features are included. In the present application, with respect to a numerical interval (i.e. a numerical range), if no special instructions are given, the optional numerical distribution within the above-mentioned numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical end points. If no special instructions are given, when the numerical interval only points to the integers within the numerical interval, it includes the two end point integers of the numerical range, as well as every integer between the two end points, which in this document is equivalent to directly listing each integer, such as t being an integer selected from 1-10, which means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise indicated, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. In the present application, the temperature parameters, if not specifically limited, allow both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows fluctuations within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed. In the present application, % (w / w) and wt% both mean weight percentage, % (v / v) means volume percentage, and % (w / v) means mass volume percentage. All documents referred to in the present application are incorporated by reference in the present application as if each document were individually incorporated by reference. Unless and to the extent that the application purpose and / or technical solution of the present application is in conflict with the cited documents, the cited documents are incorporated by reference in their entirety and for all purposes. When the present application refers to the cited documents, the definitions of the relevant technical features, terms, nouns, phrases, etc. in the cited documents are also incorporated by reference. When the present application refers to the cited documents, the examples and preferred modes of the relevant technical features cited are also incorporated by reference into the present application, but are limited by the ability to implement the present application. It should be understood that when the cited content conflicts with the description in the present application, the present application is given priority or is modified adaptively according to the description in the present application. Unless otherwise indicated, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes VII, published by Oxford University Press, 1999; Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references. The present application provides a method for stabilizing RSV F0 in the pre-F state by amino acid mutation, and provides a disulfide bond mutation position not involved in the prior art or existing patents. The position involves introducing a disulfide bond mutation between α3 / β3 of the RSV F protein, and the mutation combination based on this mutation can bind with specific monoclonal antibodies recognizing the site, and has good stability, so that the multimer of the mutant constructed not only significantly improves the expression amount of the pre-F protein, but also has good immunogenicity, can induce high levels of neutralizing antibodies, and has great potential for developing vaccines. Compared with the conventional technology, the present application finds another disulfide bond mutation position, mainly the disulfide bond mutation between α3 and β3, which is not involved in the prior art or existing patents. The single disulfide bond mutation of this position or the mutant combined with mutation of other positions can bind with specific monoclonal antibodies recognizing the site, and has good stability, so that the multimer of the mutant constructed not only significantly improves the expression amount of the pre-F protein, but also has good immunogenicity, can induce high levels of neutralizing antibodies, and has great potential for developing vaccines. The scheme of the embodiments of the present application includes: In a first aspect of the embodiments of the present application, the embodiments of the present application provide a mutant of RSV pre-fusion F protein, wherein the α3 helix region of the F1 polypeptide has a cysteine substituent 1, and the β3 sheet region has a cysteine substituent 2, and the cysteine substituent 1 and the cysteine substituent 2 form a disulfide bond. Substituent, amino acid substitution: one amino acid in an antigen is replaced by another amino acid or deletion of an amino acid. For example, an amino acid in an antigen is replaced by an amino acid from a homologous protein. Homologous protein: a protein having similar structure and function, e.g., from two or more species or viral strains having similar structure and function in the two or more species or viral strains. For example, an RSV F protein from RSV A is a homologous protein to an RSV F protein from bovine RSV. Homologous proteins share similar protein folding characteristics and can be considered structural homologs. Homologous proteins often share a high degree of sequence conservation, e.g., at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence conservation, and a high degree of sequence identity, e.g., at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. There are several subtypes of RSV, including human subtype A, human subtype B, and bovine subtype. Within RSV subtypes, there are individual strains of each subtype. In embodiments of the present application, in the mutant of the RSV pre-fusion F protein, the amino acid sequence of the remaining fragments, in addition to the F1 polypeptide forming disulfide bond through cysteine substitution 1 and cysteine substitution 2, can be wild-type sequence, or sequence with other mutations introduced through artificial or natural means, and the introduction of other mutations still enables the mutant to be stable. In some embodiments of the present application, the F1 polypeptide of the RSV pre-fusion F protein has the amino acid sequence set forth in SEQ ID NO. 51 or has at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology to the amino acid sequence set forth in SEQ ID NO. 51. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology to the amino acid sequence set forth in SEQ ID NO. 51. Alternatively, the F1 polypeptide of the RSV pre-fusion F protein has the amino acid sequence set forth in SEQ ID NO. 51, SEQ ID NO. 74, or SEQ ID NO. 75. Alternatively, The F1 polypeptide of the RSV pre-fusion F protein satisfies one or more of the following conditions: (1) does not contain a transmembrane domain, and (2) does not contain an intracellular domain. Further alternatively, the F1 polypeptide of the RSV pre-fusion F protein has one or more of the following mutations: I379V and M447V. For example, the F1 polypeptide of the RSV pre-fusion F protein has the amino acid sequence set forth in SEQ ID NO. 52. In some embodiments of the application, the distance between the cysteine substitution group 1 and the cysteine substitution group 2 is (e.g., 2, 3, 4, 5, 6, 7, ). In some embodiments of the application, the a3 helix region of the F1 polypeptide of the mutant has one or more of the following mutations: E163C, K166C, I167C, A170C, and L171C. In some embodiments of the application, the b3 sheet region of the F1 polypeptide of the mutant has one or more of the following mutations: A177C, V179C, and L181C. In some embodiments of the application, the F1 polypeptide of the mutant has one set of the following combinations of mutations: Set 1 is E163C and L181C, Set 2 is K166C and V179C, Set 3 is I167C and V179C, Set 4 is A170C and V179C, Set 5 is A170C and A177C, and Set 6 is L171C and A177C. In some embodiments of the application, the mutant further has one or more of the following mutation sites: S55C, S155C, L188C, S190F, V207L, and S290C. S55C is located in the F2 polypeptide. S155C, S180C, S186C, L188C, S190F, V207L, and S290C are located in the F1 polypeptide. In some embodiments of the application, the mutant has one set of the following combinations of mutation sites: Set 1) S155C and S290C, Set 2) S55C and L188C, and Set 3) S190F and V207L. In some embodiments of the application, the mutant does not contain the furin cleavage site fragment. In some embodiments of the application, the mutant does not contain the pep27 polypeptide, and the C-terminus of the F2 polypeptide and the N-terminus of the F1 polypeptide are directly connected by an amide bond or indirectly connected by a flexible short peptide. The term "linking" can refer to making two molecules into one continuous molecule; for example, linking two other polypeptides into one continuous polypeptide, or covalently linking a carrier molecule or other molecule to an immunogenic polypeptide, such as a mutant disclosed herein. The linking can be by chemical or recombinant means. In some embodiments of the application, optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG, or GGGGS. In some embodiments of the application, the C-terminus of the F1 polypeptide of the mutant is linked to a tag fragment and / or an aggregation motif. In some embodiments of the application, the tag fragment comprises a 6His polypeptide. In some embodiments of the application, the aggregation motif is set forth in SEQ ID NO. 48. In some embodiments of the application, the F2 polypeptide of the mutant has an amino acid sequence set forth in SEQ ID NO. 49 or has at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology to the amino acid sequence set forth in SEQ ID NO. 49. Optionally, the F2 polypeptide of the mutant has an amino acid sequence set forth in SEQ ID NO. 49, SEQ ID NO. 76, or SEQ ID NO. 77. Further optionally, the F2 polypeptide of the mutant satisfies one or more of the following conditions: 1) the C-terminus does not contain NN, and 2) has the following mutation: P102A. For example, the F2 polypeptide of the mutant has an amino acid sequence set forth in SEQ ID NO. 50. In some embodiments of the application, the mutant has an amino acid sequence set forth in any one of SEQ ID NO. 12 to SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42 to SEQ ID NO. 47, and SEQ ID NO. 53 to SEQ ID NO. 73. The mutant of the RSV pre-fusion F protein provided in the embodiments of the application has a stable conformation and can induce an immune response in a subject as an antigen / immunogen to produce antibodies. A subject is an animal. An animal is a living multicellular vertebrate or invertebrate organism, a category that includes, for example, mammals. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, such as non-human primates. Thus, administration to a subject can include administration to a human subject. Non-limiting examples of veterinary subjects include domestic animals (e.g., cats and dogs), farm animals (e.g., cows, horses, pigs, sheep, and goats), and laboratory animals (e.g., mice, rabbits, rats, hamsters, guinea pigs, and non-human primates). Antibody: A polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes or fragments thereof which specifically binds and recognizes an analyte (e.g., an antigen or immunogen), such as a RSV F protein or an antigenic fragment thereof. The immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. The term "antibody" as used herein includes antibody fragments, e.g., produced by the modification of whole antibodies and those synthesized de novo using recombinant DNA methods. Antibodies exist, for example, in the form of intact immunoglobulins and in a variety of well-characterized antibody fragments. For example, Fab, Fv, and single-chain Fv (SCFv) that bind to RSV F protein will be RSV F protein-specific binding agents. This includes intact immunoglobulins and variants and portions thereof well known in the art, such as Fab' fragments, F(ab)'2 fragments, single-chain Fv proteins ("scFv"), and disulfide knot stabilized Fv proteins ("dsFv"). A scFv protein is a fusion protein in which the light chain variable region of an immunoglobulin is joined with the heavy chain variable region of an immunoglobulin by a linker, while in a dsFv the chains have been mutated to introduce disulfide bonds to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997. Antibody fragments are defined as follows: (1) Fab, a fragment of an antibody molecule that contains the entire light chain and a portion of one heavy chain that is produced, for example, by digestion with the enzyme papain to yield an intact light chain and one heavy chain that is one portion of the original molecule; (2) Fab', a fragment of an antibody molecule that is obtained, for example, by treating whole antibody with the enzyme pepsin, followed by reduction to yield two Fab' fragments; two antibody molecules give two Fab' fragments; (3) (Fab')2, a fragment of an antibody that is obtained, for example, by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments that are held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain linked together by a short peptide linker that is expressed as a single chain. Generally, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains that are interconnected by disulfide bonds. There are two types of light chain, lambda (l) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activities of an antibody molecule: IgM, IgD, IgG, IgA and IgE. The disclosed antibodies can be class switched. Each heavy and light chain contains a constant region and a variable region, (the regions are also referred to as "domains"). In several embodiments, the heavy and light chain variable domains combine to specifically bind an antigen. In other embodiments, only the heavy chain variable domain is required. For example, naturally occurring camelid antibodies, which consist of only heavy chains, are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). The light and heavy chain variable domains contain "framework" regions interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs") (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space. CDRs are primarily responsible for binding to epitopes of antigens. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (“Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991; “Kabat” numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 27: 55-77, 2003; “IMGT” numbering scheme). The CDRs for each chain are typically referred to as CDR1, CDR2, and CDR3 (from N to C), and are usually defined by specific CDRs. The CDRs are identified by the position of the chain. Therefore, VHCDR3 is located in the variable domain of the heavy chain of the antibody in which it is contained, while VLCDR1 is a CDR1 derived from the variable domain of the light chain of the antibody in which it is contained. Light chain CDRs are sometimes referred to as CDRL1, CDRL2, and CDRL3. Heavy chain CDRs are sometimes referred to as CDRH1, CDRH2, and CDRH3. Antigen: A compound, composition, or substance that can stimulate the production of an antibody or T-cell response in an animal, including compositions injected or absorbed into the animal. An antigen is a product that reacts with a specific humoral or cellular immune response, including those induced by heterologous antigens, such as the disclosed mutant of the RSV pre-fusion F protein. Examples of antigens include (but are not limited to) polypeptides, peptides, lipids, polysaccharides, combinations thereof (e.g., glycopeptides), and nucleic acids containing antigenic determinants, such as those recognized by immune cells. In some instances, antigens include peptides derived from the pathogen of interest, such as RSV. In specific instances, antigens are derived from RSV, such as antigens comprising modified RSVF proteins stabilized in a pre-fusion conformation. An "epitope" or "antigenic determinant" refers to an antigenic region that reacts with B and / or T cells. Immunogen: A protein or portion thereof capable of inducing an immune response in mammals, such as mammals infected with or at risk of infection with pathogens. Administration of an immunogen can result in protective and / or active immunity against the pathogen of interest. Examples include the PreF mutant provided in the embodiments of this application. Immune response: the response of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus. In one embodiment, the response is specific for a particular antigen ("antigen-specific response"). In one embodiment, the immune response is a T cell response, e.g., a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response and results in the production of specific antibodies. One of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a sequence, which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence, are conserva¬tive variations wherein each resulting peptide differs in minor changes from the reference peptide. Conservative amino acid substitutions are provided in Table A below. Table A: Conservative Amino Acid Substitutions

[0182] 6) phenylalanine (F), tyrosine (Y), tryptophan (W). Not all residue positions within a protein will tolerate "conservative" substitutions. For example, if an amino acid residue is essential to the function of the protein, even a conservative substitution can destroy such activity, e.g., the specific binding of an antibody to an epitope of interest can be destroyed by a conservative mutation in the epitope. Epitope: antigenic determinant. These are specific chemical groups or peptide sequences on an antigenic molecule that cause them to induce a specific immune response, e.g., an epitope is a region of an antigen that reacts with B and / or T cells. Antibodies bind to specific antigenic epitopes, e.g., epitopes of the RSV F protein, e.g., the D25 or AM22 epitopes present on the prefusion conformation of the RSV F protein. Epitopes can be formed by contiguous amino acids or noncontiguous amino acids brought into proximity by the tertiary folding of the protein. Epitopes formed by contiguous amino acids are usually maintained in exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes usually include at least 3 and more usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining the spatial conformation of an epitope include, e.g., x-ray crystallography and nuclear magnetic resonance. Epitopes can also include post-translational modifications of amino acids, e.g., N-linked glycosylation. A "target epitope" is a particular epitope on an antigen that specifically binds to an antibody of interest, e.g., a monoclonal antibody. In some examples, a target epitope includes amino acid residues that contact an antibody of interest such that the target epitope can be selected by determining the amino acid residues that contact the antibody of interest. Amino acids in peptides, polypeptides, or proteins are typically chemically bonded together via amide bonds (CONH). In addition, amino acids can be bonded together by other chemical bonds. For example, the linkage of amino acids or amino acid analogs can include CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO- (these and others are found in Spatola, in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, ed., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, No. 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci 463-468, 1980; Hudson et al., Int J Pept Prot Res 14:177-185, 1979; Spatola et al., Life Sci 38:1243-1249, 1986; Harm J. Chem. Soc Perkin Trans. 1 307-314, 1982; Almquist et al., J. Med. Chem. 23:1392-1398, 1980; Jennings-White et al., Tetrahedron Lett 23:2533, 1982; Hollada V et al., Tetrahedron. Lett 24:4401-4404, 1983; and Hruby Life Sci 31 :189-199, 1982). Peptide Modifications: Peptides, such as the pre-fusion conformation stabilizing mutants of the embodiments of the application, can be modified, for example, to include amino acid substitutions compared to the native RSV protein sequence, or by various chemical techniques to produce derivatives having substantially the same activity and conformation as the unmodified peptide and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxy-terminal or side chain, can be provided in salt form with a pharmaceutically acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of the formula NR1R2, where R1and R2are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups of the peptide, whether amino-terminal or side chain, can be in the form of a pharmaceutically acceptable acid addition salt such as HC1, HBr, acetate, benzoate, toluenesulfonate, maleate, tartrate, and other organic salts, or can be modified to C1-C16 alkyl or dialkylamino groups or further converted to amides. Peptide Modifications: Peptides, such as the pre-fusion conformation stabilizing mutants of the embodiments of the application, can be modified, for example, to include amino acid substitutions compared to the native RSV protein sequence, or by various chemical techniques to produce derivatives having substantially the same activity and conformation as the unmodified peptide and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxy-terminal or side chain, can be provided in salt form with a pharmaceutically acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of the formula NR1R2, where R1and R2are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups of the peptide, whether amino-terminal or side chain, can be in the form of a pharmaceutically acceptable acid addition salt such as HC1, HBr, acetate, benzoate, toluenesulfonate, maleate, tartrate, and other organic salts, or can be modified to C1-C16 alkyl or dialkylamino groups or further converted to amides. The hydroxyl groups of the peptide side chains can be converted to C1-C16 alkoxy groups or C1-C16 esters using art-recognized techniques. The phenyl and phenol rings of the peptide side chains can be substituted with one or more halogen atoms such as F, CI, Br, or I or with C1-C16 alkyl groups, C1-C16 alkoxy groups, carboxylic acids and esters thereof or amides of these carboxylic acids. The methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylene groups. The thiols can be protected with any of a variety of art-recognized protecting groups such as an acetamide group. In a second aspect of the embodiments of the present application, the embodiments of the present application provide a nucleic acid molecule encoding the mutant of the RSV pre-fusion F protein of the first aspect. Nucleic acid molecule, nucleic acid: a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked by phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include synthetic non-naturally occurring analogs such as, and without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. These polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" generally refers to a short polynucleotide, typically no greater than about 50 nucleotides. It is understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes the RNA sequence (i.e., A, U, G, C) in which "U" is instead of "T." "Nucleotide" includes, but is not limited to, a monomer comprising a base linked to a sugar (e.g., a pyrimidine, purine, or synthetic analog thereof) or a monomer comprising a base linked to an amino acid (as in a peptide nucleic acid (PNA)). A nucleotide is a monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes. Thus, for example, if a gene encodes a protein, then the DNA sequence of the gene is said to encode not only the mRNA, but also the protein. As used herein, the term "coding strand" refers to the nucleotide sequence of a polynucleotide that is identical to the mRNA sequence and is used as the template for transcription. The term "non-coding strand" refers to the nucleotide sequence of a polynucleotide that is complementary to the mRNA sequence and is not used as the template for transcription. Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences which are degenerate versions of each other and which encode the same amino acid sequence. Nucleotide sequences which encode proteins and RNA can include introns. In view of degenerate variants and conservative variants, the present application does not specifically limit nucleic acid molecules. Degenerate variants and conservative variants: Polynucleotides which encode polypeptides comprising sequences which are degenerate as a result of the genetic code. For example, polynucleotides which encode the disclosed antigens or antibodies which specifically bind the disclosed antigens. There are 20 naturally occurring amino acids, most of which are specified by more than one codon. Thus, all degenerate nucleotide sequences which encode the same antigen or antigen-binding antibody are included. As a result of degeneracy in the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position in a protein coding sequence where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations" and are of a kind that do not alter the polypeptide encoded by the nucleic acid. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each "silent variation" of a nucleic acid which encodes a polypeptide is implicit to each described sequence. In some examples, which are codon-optimized for expression in a cell of a mammal, are operably linked to a promoter. Expression control sequence: A nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence that is operatively linked. An expression control sequence is operatively linked to a nucleic acid sequence when it controls and regulates transcription and, when appropriate, translation of the nucleic acid sequence. Therefore, an expression control sequence may include a suitable promoter, enhancer, transcription terminator, start codon (ATG) preceding a protein-coding gene, splicing signals for introns, maintaining the appropriate reading frame of the gene to allow proper translation of the mRNA, and a stop codon. The term "control sequence" is intended to include, at a minimum, components whose presence can affect expression, and may also include other components whose presence is advantageous, such as leader sequences and fusion chaperone sequences. An expression control sequence may include a promoter. A promoter is the smallest sequence sufficient to guide transcription. It also includes sequences sufficient to enable promoter-independent gene expression with respect to cell type specificity and histological characteristics. Those promoter elements that are tissue-specific and controllable or can be induced by external signals or agents; these elements may be located in the 5′ or 3′ region of a gene. This includes constitutive and inducible promoters (see, for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in a bacterial system, inducible promoters such as phage λ, plac, ptrp, ptac (ptrp-lac heterozygous promoter), etc., can be used. In one embodiment, when cloning in a mammalian cell system, promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from the genome of mammalian viruses (e.g., retroviral long terminal repeat sequences; adenovirus late promoters; vaccinia virus 7.5K promoters) can also be used to provide transcription of nucleic acid sequences. Polynucleotides can be inserted into expression vectors containing promoter sequences that promote efficient transcription of the inserted genetic sequence in the host. These expression vectors typically contain an origin of replication, a promoter, and specific nucleic acid sequences that allow phenotypic selection in transformed cells. RSV F proteins from different RSV subgroups, as well as nucleic acid sequences encoding these proteins and methods for manipulating and inserting these nucleic acid sequences into vectors are disclosed herein and known in the art (see, e.g., Tan et al., PLOSone, 7: e51439, 2011; Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)). In a third aspect of the embodiments of the present application, the embodiments of the present application provide a vector comprising the nucleic acid molecule of the second aspect. In a fourth aspect of the embodiments of the present application, the embodiments of the present application provide an engineered cell expressing the mutant of the RSV pre-fusion F protein of the first aspect, or comprising the nucleic acid molecule of the second aspect, or the vector of the third aspect. Expression: The translation of a nucleic acid into a protein. The protein can be expressed and remain within the cell, become a component of the cell surface membrane, or be secreted into the extracellular matrix or culture medium. Engineered cell, host cell: A cell in which a vector can propagate and express its DNA. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny can not be identical to the parental cell since there can be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. In a fifth aspect of the embodiments of the present application, the embodiments of the present application provide a method of producing the mutant of the RSV pre-fusion F protein, comprising the steps of: Culturing the engineered cell of the fourth aspect, and isolating the mutant of the RSV pre-fusion F protein from the resulting culture supernatant. In a sixth aspect of the embodiments of the present application, the embodiments of the present application provide an immunological composition comprising the mutant of the RSV pre-fusion F protein of the first aspect or the nucleic acid molecule of the second aspect, and an immunological adjuvant. Immune adjuvants: mediators used to enhance antigenicity. Adjuvants include suspensions of antigen adsorbed onto a mineral (alum, aluminum hydroxide, or phosphate) or a water-in-oil emulsion, e.g., in which an antigen solution is emulsified in mineral oil (Freund's incomplete adjuvant), sometimes including killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibit degradation of the antigen and / or cause influx of macrophages). Immune stimulatory oligonucleotides (e.g., those including a CpG motif) can also be used as adjuvants. Adjuvants include biological molecules ("biological adjuvants"), e.g., costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-alpha, IFN-gamma, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, and toll-like receptor (TLR) agonists, e.g., TLR-9 agonists. Adjuvants are well known to those of ordinary skill in the art (see, e.g., Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007). Adjuvants can be used in combination with the disclosed PreF antigens. Alternatively, the immune adjuvant in the embodiments of the present application is one or more of an aluminum salt adjuvant, a surfactant, a polynucleotide, a lipopolysaccharide, a liposome, an oil-in-water emulsion adjuvant. For example, Alum, CpG, Alum+CpG, MF59, AS04, AS01E, etc. MF59 is a water-in-oil emulsion adjuvant, which is mainly composed of three parts: oil phase, emulsifier and auxiliary agent; the oil phase is a mixture of micro-particleized short-chain triglycerides suitable for human injection; the emulsifier is a surfactant that can mix the oil phase and water uniformly; the auxiliary agent mainly includes moisturizers and buffers such as glycerol, sorbitol, ATP, etc. AS04 adjuvant is a mixture of AS03 adjuvant and MPL adjuvant. AS03 adjuvant is a mixture of liposomes, TWEEN 80, SORBITAN three surfactants. AS01E is a nanoscale liposome solution prepared from DOPC, Chol, MPL and QS-21. The main component of MPL adjuvant is lipopolysaccharide. Immunogenic compositions (immunogenic compositions): Compositions comprising antigens that induce an immune response, such as a measurable CTL response against a virus expressing an antigen or a measurable B cell response (e.g., antibody production) against an antigen. Thus, an immunogenic composition comprises one or more antigens (e.g., peptide antigens) or epitopes. An immunogenic composition may also include one or more additional components capable of inducing or enhancing an immune response, such as excipients, carriers, and / or adjuvants. In some cases, an immunogenic composition is administered to induce an immune response that protects a subject from symptoms or illnesses induced by a pathogen. In some cases, symptoms or illnesses caused by a pathogen are prevented (or reduced or improved) by inhibiting the replication of the pathogen after the subject has been exposed to it (e.g., RSV). In one example, an “immunogenic composition” includes… This includes recombinant RSVF proteins stabilized in their pre-fusion conformation, which induce measurable CTL responses against viruses expressing RSVF proteins, or induce measurable B cell responses against RSVF proteins (e.g., antibody production). It further refers to isolated nucleic acids encoding antigens, such as nucleic acids that can be used to express antigens (and thus to induce immune responses against such peptides). For in vitro use, the immunogenic composition may include an antigen or a nucleic acid encoding an antigen. For in vivo use, the immunogenic composition will typically include a protein, immunogenic peptide, or nucleic acid in a pharmaceutically acceptable carrier and / or other pharmaceutical agent. The ability of any particular peptide, such as a disclosed RSVF protein stabilized in its pre-fusion conformation or a nucleic acid encoding a disclosed RSVF protein stabilized in its pre-fusion conformation, to induce a CTL or B cell response can be readily tested using recognized assays. The immunogenic composition may include adjuvants well known to those skilled in the art. Immunological reaction conditions: These include conditions that allow antibodies generated against a specific epitope to bind to said epitope and to a degree that is detectably greater than that of substantially all other epitopes and / or substantially exclude binding to substantially all other epitopes. Immunological reaction conditions depend on the form of antibody binding reaction and are generally those used in immunoassay protocols or encountered in vivo. The immunological reaction conditions used in the methods are “physiological conditions,” which include references to typical conditions (e.g., temperature, molar osmolality, pH) within living mammals or mammalian cells. While some organs are known to experience extreme conditions, the in vivo and intracellular environment is typically around pH 7 (e.g., pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the primary solvent, and exists at temperatures above 0°C and below 50°C. Molar osmolality is within the range supporting cell viability and proliferation. In a seventh aspect of the embodiments of the present application, the embodiments of the present application provide use of the mutant of the pre-fusion F protein of RSV in the first aspect in the preparation of a respiratory syncytial virus antibody detection kit. In the embodiments of the present application, the respiratory syncytial virus antibody detection kit can be used for detecting the corresponding antibody, and the definition of the antibody refers to the first aspect, which can be a neutralizing antibody or a binding antibody. In an eighth aspect of the embodiments of the present application, the embodiments of the present application provide a respiratory syncytial virus antibody detection kit, which comprises the mutant of the pre-fusion F protein of RSV in the first aspect. The definition of the respiratory syncytial virus antibody detection kit refers to the seventh aspect. In a ninth aspect of the embodiments of the present application, the embodiments of the present application provide a method for preventing and treating lower respiratory tract infection caused by respiratory syncytial virus, which comprises the following steps: administering a therapeutically effective amount of the immunological composition in the sixth aspect to a subject. Administration: introduction of a composition into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intravenous, the composition is administered by introducing it into the subject's vein. Effective amount: the amount of an agent, such as a PreF antigen or a nucleic acid encoding a PreF antigen, or other agent, sufficient to produce a desired response, such as an immune response to RSV F protein, or to reduce or eliminate signs or symptoms of a condition or disease, such as RSV infection. For example, this can be the amount required to inhibit viral replication or measurably alter outward symptoms of viral infection. Generally, such an amount will be sufficient to measurably inhibit viral (e.g., RSV) replication or infectivity. When administered to a subject, a dose will typically be used that will achieve a target tissue concentration (e.g., in respiratory tissue) that has been demonstrated to achieve in vitro inhibition of viral replication. In some examples, an "effective amount" is an amount that treats (including prophylaxis) one or more symptoms and / or underlying cause of any condition or disease, such as treating RSV infection. In one example, an effective amount is a therapeutically effective amount. In one example, an effective amount is an amount that prevents development of one or more signs or symptoms of a particular disease or condition (e.g., one or more signs or symptoms associated with RSV infection). "Prevention" refers to the suppression of the full development of a disease or symptom in subjects at risk of developing a disease (e.g., RSV infection). "Treatment" refers to a therapeutic intervention that improves the signs or symptoms of a disease or pathological condition after it has begun to develop. The term "improvement" in relation to a disease or pathological condition refers to any observable beneficial therapeutic effect. A beneficial effect can be demonstrated, for example, by the delayed onset of clinical symptoms of the disease in susceptible subjects, a reduction in the severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or condition of the subject, or other parameters known in the art to be specific to a particular disease. "Prophylactic" treatment is a treatment administered to subjects who do not exhibit signs of disease or only exhibit early signs in order to reduce the risk of developing the lesion. The term "reduction" is relative, meaning that a drug reduces a reaction or symptom if the reaction or symptom is quantitatively reduced after administration, or if it is reduced compared to a reference drug after administration. Similarly, the term "prevention" does not necessarily mean that the drug completely eliminates a reaction or symptom, provided that at least one characteristic of the reaction or symptom is eliminated. Therefore, immunogenic compositions that reduce or prevent infection or reaction (e.g., pathological reaction, such as a vaccine-enhanced viral disease) can, but do not necessarily, completely eliminate such infection or reaction, provided that the infection or reaction is measurably reduced compared to an infection or reaction in the absence of the agent or compared to a reference agent, for example, by at least about 50%, such as at least about 70%, or about 80%, or even about 90% (i.e., reduced to 10% or less). In a tenth aspect of this application, an embodiment of this application provides a method for detecting or separating RSVF-binding antibodies in a sample. The method includes the following steps: The mutant of the RSV pre-fusion F protein described in the first aspect is contacted with an RSVF-binding antibody in the sample to form an immune complex; and, The immune complex is detected to detect or isolate RSV F-binding antibodies in a sample. The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art. In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision. Example 1 1. Design of RSV pre-F mutants This example relates to the structure of the F0 protein of respiratory syncytial virus, as shown in Figure 1. Figure 1 depicts the structure of the wild-type RSV viral F protein precursor polypeptide, corresponding to the amino acid sequences of SEQ ID NO. 1 (human wild-type RSV A, STRAIN A2, 574 aa), SEQ ID NO. 2 (human wild-type RSV B, STRAIN 18537, 574 aa) and SEQ ID NO. 3 (bovine wild-type RSV B, STRAIN A51908, 572 aa). In the case of the native full-length human RSV A2, the precursor polypeptide comprises a signal peptide (aa 1-25, as shown in SEQ ID NO. 7), an F2 polypeptide (aa 26-109, as shown in SEQ ID NO. 5), a pep27 polypeptide (aa 110-136, as shown in SEQ ID NO. 6), an Fl polypeptide (aa 137-574, as shown in SEQ ID NO. 4), and the furin cleavage sites RARR and KKRKRR. The structure of the native full-length human RSV B is consistent with that of the native full-length human RSV A2. In the case of the native full-length human RSV A2, the precursor polypeptide comprises a signal peptide (aa 1-25, as shown in SEQ ID NO. 7), an F2 polypeptide (aa 26-109, as shown in SEQ ID NO. 5), a pep27 polypeptide (aa 110-136, as shown in SEQ ID NO. 6), an Fl polypeptide (aa 137-574, as shown in SEQ ID NO. 4), and the furin cleavage sites RARR and KKRKRR. The structure of the native full-length human RSV B is consistent with that of the native full-length human RSV A2. The a3 helix in the native full-length human RSV A2 precursor polypeptide, the human RSV B precursor polypeptide, and the native full-length bovine RSV B precursor polypeptide are shown in SEQ ID NO. 8, SEQ ID NO. 53, and SEQ ID NO. 53, respectively, and the b3 sheet in the three native full-length precursor polypeptides is shown in SEQ ID NO. 9. Table 1 The native RSV F protein is first expressed as an F0 polypeptide (precursor). The F0 polypeptide is processed by intracellular furin-like proteases at two sites after the endoplasmic reticulum translocation signal peptide is cleaved, and the pep27 polypeptide is removed, and the mature F protein comprises an F2 polypeptide at the N-terminus and an Fl polypeptide at the C-terminal portion, which is linked to the F2 polypeptide by two disulfide bonds. The mature F protein forms a trimer and is anchored to the cell membrane via the Fl polypeptide through the transmembrane domain. Taking the native full-length human RSV precursor polypeptide as an example, the design of the pre-F mutant of the RSV F protein is shown in Figures 2 and 3: (1) deleting the P27 polypeptide and using GS as a linker to connect the Fl and F2 polypeptides; (2) deleting the transmembrane domain and intracellular domain of the protein, so that the mutant achieves soluble expression; (3) introducing a trimer structure motif at the C-terminal of the monomeric protein of the mutant, so that the mutant is expressed and secreted in the form of a trimer; (4) adding a His tag at the C-terminal end to facilitate purification and preparation of the mutant. The RSV F protein undergoes a conformational change from pre-F conformation to Post F conformation, and the N-terminal and C-terminal of the F1 polypeptide are conformationally changed into an upward long alpha helix. By analyzing its structure, it is found that introducing a disulfide bond between its alpha3 helix and beta3 sheet can obtain a stabilized pre-F conformation. Table 2 Figure 2 describes a structural schematic diagram of a stabilized mutant RSV F protein polypeptide monomer for construction (the amino acid sequence is SEQ ID NO. 10). The schematic diagram corresponds to the natural mutant of RSV / A2 subtype (the amino acid sequence is SEQ ID NO. 1), and the positions of the three natural mutations are P102A, I379V and M447V, respectively. Unless otherwise specified, other mutations involved in this application are designed on this basis. The mutants involved in the implementation cases in this application are all based on amino acid mutations. Unlike the wild-type F protein, the pep27 polypeptide is replaced by GS, which is not affected by furin, the transmembrane domain is deleted, and a His-Tag is added at the C-terminal end of the protein to facilitate purification. After expression of such mutants, they exist in the form of soluble monomers in the cell culture supernatant. Figure 3 describes a structural schematic diagram of a stabilized mutant RSV F protein polypeptide trimer for construction (the amino acid sequence is SEQ ID NO. 11). Unlike the wild type, the pep27 peptide is replaced by GS, which is not affected by furin. Preferably, the mutant RSV F protein of the present application forms a trimer to simulate the natural trimer state of the F protein, and therefore, the trimerization motif (T4 foldon) of the bacteriophage T4 fiber protein (foldon) is fused to the C-terminal end, and a purification tag is added at the C-terminal end. After expression of such mutants, they exist in the form of soluble trimers in the cell culture supernatant. Figure 4 describes that the natural RSV F0 precursor protein or mature F protein coexists with 7 pairs of disulfide bonds, which play an important role in stabilizing the structural characteristics of the RSV F protein. Among them, the F2 polypeptide and the F1 polypeptide are connected together by two pairs of disulfide bonds (C37 / C439; C69 / C212), which are important for stabilizing the RSV pre-F vertex. Epitopes play a key role. The remaining 5 disulfide bonds are distributed in the F1 polypeptide to facilitate the unique structural features of the F protein. The present application mutates other positions based on maintaining the native disulfide bonds of the RSV F protein, and maintains the pre-F conformation without changing the basic structure of the RSV F protein. Table 3, native disulfide bond pairing of wild-type RSV F0 protein Figure 5 describes a schematic diagram of the three-dimensional structure of the RSV F protein monomer (left side) and a local enlarged schematic diagram of the a3 and b3 sheets (right side). The schematic diagram shows that the a3 and b3 sheets are anti-parallel and close in distance. The RSV pre-F conformation is metastable, and an irreversible rearrangement occurs to mediate membrane fusion or a non-functional post-F conformation when triggered spontaneously. Among them, the a2, a3, b3, b4, a4 and a5 in the sequence are configured into a long coiled coil bundle, which is a typical feature of the post-F. Therefore, preventing its conformation change by amino acid mutation is an important means to maintain the stable pre-F conformation. In the present application, we found that the disulfide bond formed by suitable amino acid mutations between the a3 helix and the b3 sheet can produce a stabilized pre-F conformation, and the combination of mutations formed on this basis can further improve the stability of the pre-F mutant. Table 4, amino acids contained in the a3 helix and the b3 sheet of the RSV F0 protein Figure 6 describes the amino acids involved in the a3 and b3 positions in the present application and the interatomic distance between the two amino acids prone to form disulfide bonds Table 5, mutation sites of the a3 helix and the b3 sheet and other regions in each mutant The " / " in the table indicates that the mutations S55C, S155C, S180C, S186C, L188C, S190F, V207L and S290C are not contained. 2. Vector construction of RSV pre-F mutants According to the mutation strategy to determine the amino acid sequence of the mutant, the nucleic acid sequence corresponding thereto is codon-optimized, and the optimized codon facilitates expression in Chinese hamster ovary cells Cricetulus griseus (CHO cells). Finally, it is connected to the pcDNA3.1 vector through BamH I and Xho I enzyme digestion sites. The substitution, insertion and deletion of vectors were performed using ClonExpress II Recombinant Cloning Kit (Novagen). The fragments at both ends of the mutation site were amplified by polymerase chain reaction (PCR) with high-fidelity enzyme Phanta Max (Novagen), and the two fragments were fused by PCR after gel recovery. The fused fragments were homologously recombined into the pcDNA3.1 vector digested by BamH I and Xho I. The constructed point mutation vector was sequenced by Beijing Genesee Biotech Co., Ltd. and confirmed to have no error in the sequence. The cloning vector was inoculated into 300 mL of LB (Amp+) medium and cultured at 37°C and 180 rpm for 16 h. The plasmid was extracted using a large-scale / plasmid extraction kit, and finally stored in 1 mL of sterile TE buffer. For all commercial kits or reagents, the manufacturer's instructions were followed. 3. Expression of RSV pre-F mutants The mutant proteins were expressed by ExpiCHOTM expression system (Thermofisher). The transient expression was performed according to the manufacturer's standard protocol. Briefly, one day before transfection (day -1), the ExpiCHO-STM culture was split, and the final density was 3 x 10 6 - 4 x 10 6 live cells / mL. The next day (day 0), the viable cell density and the percentage of viability were determined. The cell density should reach about 7 x 10 6 - 10 x 10 6 live cells / mL. The viability should be 95-99%, and the transfection could continue. The cells were diluted to a final density of 6 x 10 6 live cells / mL using fresh preheated ExpiCHOTM expression medium at 37°C. The flask was gently shaken to mix the cells, and the remaining cells were discarded. The ExpiFectamineTM CHO / plasmid DNA complex was prepared according to the reagent instruction using cold reagents (4°C). After inversion and mixing, the ExpiFectamineTM CHO / DNA complex was added to the flask, and the cells were cultured on a shaker (8% CO2, 37°C, 120 rpm). The next day after transfection (day 1, 18-22 hours after transfection), ExpiFectamineTM CHO enhancer and ExpiCHOTM supplement were added. The culture was terminated on the 8th day after transfection according to the standard test protocol, and samples were taken for detection. 4. Purification of RSV pre-F mutants Protein purification was performed in two steps using affinity chromatography and ion exchange chromatography. In summary, the expression product was harvested by centrifugation (8000 rpm, 20 min) to remove cells and cell debris from the culture supernatant. The culture supernatant was filtered through a 0.45 pm filter to remove impurities. The treated supernatant was applied to a Ni-Sepharose 6 FF column (cytiva) equilibrated with 25 mM Tris-HCl, 0.15 M NaCl (Buffer A; pH 8.0). The column was then washed with 25 mM Tris-HCl, 150 mM NaCl, pH 8.0 until A280 reached baseline level, and eluted with a linear gradient of elution buffer (Buffer B: 25 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 8.0) and the eluate was collected. The imidazole was removed by using equilibration buffer as a displacement liquid. After the change, the sample was diluted with PBS buffer to a conductivity of less than 4 ms / cm and the pH was adjusted to 6.0. The protein was applied to a Capto S Impres packing (Cytiva) equilibrated with 20 mM phosphate buffer, pH 6.0. After the application was complete, the column was washed with 20 mM phosphate buffer, pH 6.0 until A280 reached baseline level, and eluted with a linear gradient of elution buffer (Buffer B: 20 mM phosphate buffer, 1 M NaCl, pH 6.0) and the eluate was collected. The pH was adjusted to 7.4-8.0 with 0.2 M Na2HP04, and the eluate was further filtered through a 0.45 pm filter to remove insoluble particles. After the change, the sample was diluted with PBS buffer to a conductivity of less than 4 ms / cm and the pH was adjusted to 6.0. The protein was applied to a Capto S Impres packing (Cytiva) equilibrated with 20 mM phosphate buffer, pH 6.0. After the application was complete, the column was washed with 20 mM phosphate buffer, pH 6.0 until A280 reached baseline level, and eluted with a linear gradient of elution buffer (Buffer B: 20 mM phosphate buffer, 1 M NaCl, pH 6.0) and the eluate was collected. The pH was adjusted to 7.4-8.0 with 0.2 M Na2HP04, and the eluate was further filtered through a 0.45 pm filter to remove insoluble particles. 5. SDS-PAGE identification of RSV pre-F mutants The purified recombinant protein was detected by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). The detection sample after purification was added to a sample buffer containing SDS and a reducing agent (such as dithiothreitol or mercaptoacetic acid), treated with boiling water, and then applied to the gel. After electrophoresis, the gel was stained with Coomassie blue, and the gel image was obtained using a transparent scanner or a protein imaging system. Figure 7 depicts the SDS-PAGE identification results of mutant monomer and trimer based on the position mutation of a3 / b3. Since the mutant is only amino acid substitution from wild type F protein, the molecular weight of different mutant proteins is basically the same, and the same on SDS-PAGE, so only JW-05 represents the mutant protein monomer. Similarly, JW-05-T4 represents the mutant trimer. As can be seen from the results, the monomer and trimer are slightly larger than the theoretical molecular weight (51 KDa / 54 KDa), which may be due to the glycosylation of the mutant. 6. HPLC detection of purity and uniformity of the mutant The purity of the mutant protein was analyzed by HPLC (High Performance Liquid Chromatography) using Thermo U3000 high performance liquid chromatograph and (Thermo) TSKgel UP-SW2000 molecular exclusion column (TOSOH) chromatographic column. The mobile phase is 20 mM PBS buffer, the flow rate is 1 mL / min, and the elution composition is detected at 280 nm. HPLC detects the purity and uniformity of the target protein. Figure 8 depicts the HPLC detection results of mutant monomer and trimer. Among them, Figure 8a shows that the characteristic peak of JW-05 monomer protein appears at 18.731 min. Figure 8b shows that the characteristic peak of trimer JW-05-T4 appears at 15.958 min, and the target protein is single and no protein aggregation phenomenon occurs. 7. Double antibody sandwich method for detecting the content of RSV pre-F mutant The double antibody sandwich ELISA method was used to quantify the RSV pre-F mutant. Palivizumab, which can recognize pre-F, post-F and F protein at the same time, was used as the coating antibody, and the specific recognition of RSV pre-F mutant protein was used as the detection antibody. The standard curve was drawn by using the standard protein of RSV pre-F mutant, and the content of the sample was calculated by the standard curve. The site-specific monoclonal antibody D25 labeled with horseradish peroxidase is used as the detection antibody (D25-HRP). Meanwhile, a standard curve is established using purified pre-F monomer protein and trimer protein as calibrators, respectively. The content of the sample to be tested is obtained by detecting the absorbance at OD450 and the dilution factor through the standard curve. This method can detect the expression of mutant pre-F protein under transient expression and the residual content of pre-F mutant protein in the mutant thermal stability test. The sample detection process is as follows: monoclonal antibody is diluted to 1 μg / L with carbonate buffer and coated on an enzyme-labeled plate (Corning 9018). 100 μL per well, 37°C for 1 h, then 2-8°C overnight; discard the liquid in the 96-well plate, wash 3 times with 20 mM PBS, then add 200 μL blocking solution (2% bovine serum albumin, component V) per well, block at room temperature for 60 min; aspirate the blocking solution in the wells, wash 3 times with 20 mM PBS-T solution, add the mutant pre-fusion conformation F protein to the first column of the 96-well enzyme-labeled plate after a series of 3-fold dilutions, the negative control is PBS, 37°C for 60 min, aspirate the blocking solution in the wells, wash 3 times with 20 mM PBS-T solution; take the anti-HIS-HRP conjugate, dilute it with enzyme conjugate diluent 1:2000, then add it to the 96-well enzyme-labeled plate, 100 μL per well, 37°C for 10 min; aspirate the secondary antibody in the wells, wash 3 times with 20 mM PBS-T solution, add 100 μL of TMB color developing solution per well, add 50 μL of stop solution to terminate the reaction after 10 min, then measure A450 and absorbance with an enzyme-labeled instrument. Figure 9 shows the expression of pre-F protein in the cell culture supernatant after transient transfection of monomeric RSV pre-F mutant protein in Expi-CHO cells. In this example, the results show that the natural F protein (WT) can also be detected in the culture supernatant of transient expression with relatively low pre-F protein. When the p27 peptide is deleted and replaced by GS (F0-GS, SEQ ID NO. 10), it leads to an increase in the content of pre-F protein. When the cysteine substitution is introduced into the a3 / b3 position of the F0 protein, the mutant significantly increases the content of pre-F protein due to the formation of disulfide bridges, indicating that the disulfide bond formed at the a3 / b3 position prevents the conformational change of F protein to post-F, so that the F protein maintains the pre-fusion conformation. Figure 10 shows the percentage of pre-F protein remaining after heat treatment at 50°C for 1 h, 2 h and 3 h, respectively, of purified monomeric mutant proteins at an antigen concentration of 0.1 mg / mL in a buffer system containing 20 mM PB and 150 mM NaCl at pH 7.4. The results show that WT and F0-GS, although pre-F protein can be detected, have very poor stability. Mutations at the a3 / β3 position significantly improve the stability of pre-F protein compared to WT and F0-GS. The results in Figure 11 show that disulfide bonds formed at the a3 / β3 position in combination with mutations at other positions also can form stable pre-F protein, Although there are significant differences in the pre-F protein content of the different combinations, the pre-F protein expression is reduced when trimerized with the T4 foldon motif compared to monomeric expression. The results in Figure 12 show that mutant trimers based on mutations at the a3 / β3 position have higher thermal stability than monomers, and in particular that combinations of mutations at the a3 / β3 position have better thermal stability than single a3 / β3 position disulfide bond mutations, indicating that mutations at the a3 / β3 position provide a very promising possibility for generating stabilized pre-F. The results in Figures 13 and 14 show that purified pre-F mutants can be recognized by the specific monoclonal antibodies D25 and AM22 directed against the pre-F protein in the The results in Figure 15 show that although different mutants can form pre-F trimeric protein, there are significant differences in the pre-F protein mutants at different positions when detected by the pre-F protein trimer-specific monoclonal antibody AM14. The reason is that the mutations at different positions cause subtle structural differences that result in a change in protein conformation after trimerization and are not recognized by the AM14 monoclonal antibody. AM14 recognizes only trimeric pre-F protein, and this result confirms that the trimeric pre-F mutants can assemble correctly. 8. Evaluation of immunogenicity of RSV pre-F protein To evaluate the immunogenicity of different pre-fusion conformation RSV F, mice were immunized with different mutant proteins. Female Balb / c mice, 14-16 g, were immunized with 5 μg of vaccine antigen with aluminum hydroxide as an adjuvant. The RSV inactivated vaccine (FI-RSV) group was added as a control. Intramuscular injection was performed at weeks 0 and 4 (28 d). Serum was collected 2 weeks after the second immunization (42 d) to determine total IgG antibody titers and neutralizing antibody titers. The immunization groups are shown in the table below: Table 6 ​9. Antigen-specific antibody level detection Total IgG antibody detection was performed by indirect ELISA. Pre-F protein was pre-coated in 96-well plates. After blocking, the serum to be tested was added. The serum to be tested was diluted by a factor of 800, followed by a three-fold dilution. After washing to remove unbound serum, HRP-labeled goat anti-mouse secondary antibody was added for incubation. After incubation, unbound secondary antibody was removed by washing. After washing, substrate was added for color development. The mouse serum titer was determined by detecting the absorbance at 450 nm and 630 nm. The dilution titer was calculated based on the dilution factor of the serum in the last well with an absorbance greater than the CUT-OFF value. Different mutant trimer proteins were added to BalB / c mice with aluminum hydroxide adjuvant, and inactivated vaccine was used as a control. The pre-F mutant trimer was used as a coating antigen to detect the antigen-specific IgG antibody level in mouse serum. The results in FIG. 16 show that the five pre-F mutant trimers have good immunogenicity and can induce a high level of antibody response. In particular, the mutant trimer produced by the combination of mutations based on the a3 / b3 position induces a higher antibody level than a single mutation. The reason is that the different positions of the mutant cause differences in the structure of the antigen, which in turn causes changes in the stability of the protein, and finally affects the immunogenicity of the protein. Although there are almost no antibodies against the antigen epitope of the a3 / b3 position in the inactivated vaccine, there are antibodies against other antigen epitopes in the immune serum of the inactivated vaccine. Although there are almost no antibodies against the antigen epitope of the a3 / b3 position in the inactivated vaccine, there are antibodies against other antigen epitopes in the immune serum of the inactivated vaccine. 10. Neutralizing antibody level detection Neutralizing antibody titer detection was completed by the plaque method. First, a 24-well plate was prepared with a monolayer of Hep2 cells. The serum was heated in a water bath at 56°C for 30 min. After inactivation, the serum was cooled to room temperature, and the serum was diluted with EMEM serum-free medium in proportion (1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048). 100 μL of virus liquid with a titer of 50 pfu was mixed with the serum diluent in equal amounts, and incubated at 37°C for 1 h. The culture solution in the ppC VBNM well plate was removed, and washed twice with sterile PBS. The remaining serum was removed, and the serum-virus mixture after incubation was added to the well. The incubation was continued in the incubator for 1 h, with shaking every 15 min. The serum-virus mixture was removed, and washed twice with sterile PBS. The remaining serum was removed, and the substrate was added for color development. The neutralizing antibody titer was determined by detecting the absorbance at 450 nm and 630 nm. The mixture was overlaid with 2 mL of 0.5% low-melt agar containing 2% serum, and incubated at 37°C in 5% CO2for 9 days. After 9 days, the overlay was removed, and the residual overlay was gently washed off with PBS. About 0.5 mL of cold methanol was added to fix the cells for 10 min. The methanol was discarded, and the cells were stained with crystal violet staining solution for 10 min. The crystal violet staining solution was discarded, and the cells were washed twice with deionized water. The number of plaques was counted, and the neutralizing antibody titer was defined as the dilution of serum that resulted in a 50% reduction in infectious units (NT50). Figure 17 shows the results of neutralizing antibody detection of mutant proteins and RSV inactivated vaccine (FI-RSV). It is shown that the mutant trimer has good immunogenicity and can induce high levels of neutralizing antibodies against RSV A2 in mice, and the level of neutralizing antibodies is significantly higher than that of the inactivated vaccine group. To verify the presence of neutralizing epitopes in the mutants, three healthy adult sera were screened and verified as positive sera by RSV / A2 strain neutralization test. For the three healthy adult sera, the mutant monomer antigens were adsorbed using Beaver Beads™ His-tag Protein Purification magnetic beads (BEAVER BIO). First, the magnetic beads were washed twice with PBS (pH 7.4) and divided into 50 μL aliquots. 50 μg of monomer proteins JW-04-T4, JW-05-T4, JW-25-T4, and JW-29-T4 were mixed with the magnetic beads, respectively, and then blocked with bovine serum albumin. Then, 100 μL of each healthy adult serum was diluted 10-fold with PBS (pH 7.4), and the magnetic beads after antigen adsorption were added to the serum diluent. The mixture was incubated at 4°C for 1 h under stirring, and the supernatant was collected by centrifugation to remove the antibodies bound to the antigen. The serum diluent after adsorption was subjected to virus infection cell experiments according to the operation procedure of neutralizing antibody detection. The human serum diluent without antigen adsorption was used as a positive control for virus neutralization test. The positive control should have no cytopathic effect 3-4 days after virus cell infection, and the cell state should be consistent with the blank control cell wells, indicating that the positive serum can neutralize virus infection. The virus can normally infect cells and cause typical cytopathic effect 3-4 days later, which is considered a valid test. The neutralization ability of the antigen-adsorbed serum to the virus was evaluated. Figure 18 shows the adsorption rate of neutralizing antibodies in the serum. The results show that the serum without antigen adsorption can completely neutralize the virus, and the cells do not have cytopathic effect. The serum after adsorption with each mutant antigen completely removes the neutralizing antibodies, and the serum cannot prevent virus infection of cells, resulting in typical cytopathic effect. This result shows that each mutant has the same neutralizing epitope and can be recognized by naturally infected serum antibodies, which is the basis for developing RSV pre-F candidate vaccines. Example 2 Referring to the design of JW-01 to JW-06 in Example 1, the F protein precursor polypeptides of human RSV STRAIN 18537 and bovine RSV STRAIN A51908 were used as starting points to design the corresponding mutants JW-01-18537 to JW-06-18537, JW-01-51908 to JW-06-51908, respectively, as shown in the following table. At the same time, the content of the mutants (denoted as V) was detected by the double antibody sandwich method of Example 1. Further, the samples with detected content were divided into two groups for processing (one group was placed at 4°C for 4 weeks, and the other group was placed at 50°C for 3h), and then the content of the mutants in the samples (denoted as V') was detected again, and V' / V x 100% was calculated, and the results are shown in the following table. Table 7 Note: "N / A" means no detection; "not detected" means detection but no detection. Table 7 describes the effect of introducing disulfide bonds at the α3 helix and β3 sheet of the F protein of different subtypes of RSV viruses on the expression amount and stability of the mutants. The results show that because the F proteins of different subtypes of RSV viruses are relatively conservative, introducing disulfide bonds at the α3 helix and β3 sheet has similar effects on the expression amount and stability of the F proteins of different subtypes of RSV viruses, and can express preF proteins to different degrees. Example 3 Referring to the design of JW-05 in Example 1, this example designs mutants JW-L01 to JW-L09, as shown in the following table 8. At the same time, the content of the mutants (denoted as V) was detected by the double antibody sandwich method of Example 1. Further, the samples with detected content were divided into two groups for processing (one group was placed at 4°C for 4 weeks, and the other group was placed at 50°C for 3h), and then the content of the mutants in the samples (denoted as V') was detected again, and V' / V x 100% was calculated, and the results are shown in the following table 9. Table 8, F protein p27 polypeptide different flexible short peptide mutants Table 9, expression amount and thermal stability of F protein p27 polypeptide different flexible short peptide mutants Table 9 describes the expression amount and thermal stability of the mutant proteins obtained after using different flexible short peptides to connect the p27 position of the mutant F proteins at the α3 helix and β3 sheet. The results show that there is a slight difference in the thermal stability of the mutant proteins obtained by different lengths of flexible short peptides and different connection positions, and they can all be normally expressed. Table 10 Each of the technical features of the above-described embodiments and examples can be combined in any suitable manner. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments and examples are described, however, as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure. The above-described embodiments only express several implementation manners of the present application, facilitate understanding of the technical solutions of the present application in detail, but should not be understood as a limitation on the patent protection scope of the present application. It should be noted that, for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. In addition, it should be understood that, after reading the above description of the present application, those skilled in the art can make various modifications or improvements to the present application, and the equivalent forms also fall within the protection scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

A mutant of a RSV pre-fusion F protein, the F1 polypeptide of which has a Cysteine Substitution 1 in the alpha 3 helix region and a Cysteine Substitution 2 in the beta 3 sheet region, the Cysteine Substitution 1 and the Cysteine Substitution 2 forming a disulfide bond. The mutant of RSV pre-F protein according to claim 1, wherein, The species origin of the RSV pre-fusion F protein is human or bovine. The mutant of a RSV pre-F protein according to claim 1 or 2, wherein The F1 polypeptide of the RSV pre-fusion F protein has an amino acid sequence of SEQ ID NO. 51 or has at least about 80% homology to the amino acid sequence of SEQ ID NO.

51. Optionally, the F1 polypeptide of the RSV pre-fusion F protein has an amino acid sequence of SEQ ID NO. 51, SEQ ID NO. 74, or SEQ ID NO.

75. Further optionally, the F1 polypeptide of the RSV pre-fusion F protein satisfies one or more of the following conditions: (1) does not contain a transmembrane domain, and (2) does not contain an intracellular domain. Still further optionally, the F1 polypeptide of the RSV pre-fusion F protein has one or more of the following mutations: I379V and M447V. Yet still further optionally, the F1 polypeptide of the RSV pre-fusion F protein has an amino acid sequence of SEQ ID NO.

52. The mutant of RSV pre-F protein according to any one of claims 1 to 3, wherein, the distance between the cysteine substituent 1 and the cysteine substituent 2 is about 5-15 A The mutant of RSV pre-F protein according to any one of claims 1 to 4, wherein, The alpha 3 helix region of the F1 polypeptide of the mutant has one or more of the following mutations: E163C, K166C, I167C, A170C, and L171C. The RSV pre-F protein mutant of any one of claims 1 to 5, wherein, The beta 3 sheet region of the F1 polypeptide of the mutant has one or more of the following mutations: A177C, V179C, and L181C. The mutant of RSV pre-F protein according to any one of claims 1 to 6, wherein, The F1 polypeptide of the mutant has one of the following combinations of mutations: Group 1 is E163C and L181C, Group 2 is K166C and V179C, Group 3 is I167C and V179C, Group 4 is A170C and V179C, Group 5 is A170C and A177C, and Group 6 is L171C and A177C. The mutant of RSV pre-F protein according to any one of claims 1 to 7, wherein, The mutant further has one or more of the following mutation sites: S55C, S155C, L188C, S190F, V207L, and S290C. The mutant of RSV pre-F protein according to any one of claims 1 to 8, wherein, The mutant has one of the following combinations of mutation sites: Group 1) S155C and S290C, Group 2) S55C and L188C, and Group 3) S190F and V207L. The mutant of RSV pre-F protein according to any one of claims 1 to 9, wherein, The mutant does not contain a furin cleavage site fragment. The mutant of RSV pre-F protein according to any one of claims 1 to 10, wherein, The mutant does not contain a pep27 polypeptide, the C-terminus of the F2 polypeptide and the N-terminus of the F1 polypeptide are directly connected by an amide bond or indirectly connected by a flexible short peptide; Optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG, or GGGGS. The mutant of RSV pre-F protein according to any one of claims 1 to 11, wherein, The F1 polypeptide of the mutant has one or more of the following: a C-terminal connecting tag fragment and an aggregation motif. Optionally, the tag fragment comprises a 6His polypeptide. Optionally, the aggregation motif is set forth in SEQ ID NO.

48. The mutant of RSV pre-F protein according to any one of claims 1 to 12, wherein, The F2 polypeptide of the mutant has an amino acid sequence set forth in SEQ ID NO. 49 or has at least about 80% homology to the amino acid sequence set forth in SEQ ID NO.

49. Optionally, the F2 polypeptide of the mutant has an amino acid sequence set forth in SEQ ID NO. 49, SEQ ID NO. 76 or SEQ ID NO.

77. Further optionally, the F2 polypeptide of the mutant satisfies one or more of the following conditions: 1) the C-terminus does not contain NN, and 2) has a mutation of P102A; Still further optionally, the F2 polypeptide of the mutant has an amino acid sequence set forth in SEQ ID NO.

50. The mutant of RSV pre-F protein according to any one of claims 1 to 13, wherein, The amino acid sequence of the mutant is set forth in any one of SEQ ID NO. 12 to SEQ ID NO. 35, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42 to SEQ ID NO. 47 and SEQ ID NO. 53 to SEQ ID NO.

73. A nucleic acid molecule encoding the mutant of the RSV pre-fusion F protein of any one of claims 1 to 14. A vector comprising the nucleic acid molecule of claim 15. An engineered cell expressing the mutant of the RSV pre-fusion F protein of any one of claims 1 to 14, or comprising the nucleic acid molecule of claim 15 or the vector of claim 16. A method of producing the mutant of the RSV pre-fusion F protein of any one of claims 1 to 14, comprising the step of: culturing the engineered cell of claim 17, and isolating the mutant of the RSV pre-fusion F protein from the resulting culture supernatant. An immunological composition comprising the mutant of the RSV pre-fusion F protein of any one of claims 1 to 14, or the nucleic acid molecule of claim 15, and an immunological adjuvant; Optionally, the immunological adjuvant comprises one or more of an aluminum salt adjuvant, a surfactant, a polynucleotide, a lipopolysaccharide, a liposome, an oil emulsion adjuvant. Use of the mutant of the RSV pre-fusion F protein of any one of claims 1 to 14 in the manufacture of a respiratory syncytial virus antibody detection kit. A respiratory syncytial virus antibody detection kit comprising the mutant of the RSV pre-fusion F protein of any one of claims 1 to 14. A method of preventing or treating lower respiratory tract infection caused by respiratory syncytial virus, comprising the step of administering to a subject a therapeutically effective amount of the immunological composition of claim 19.

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