RSV pre-fusion F protein mutant and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an RSV pre-fusion F protein mutant and application thereof. The RSV pre-fusion F protein mutant has better stability and higher expression quantity and / or immunogenicity, and can be used for preparing an RSV antibody, preventing RSV infection, preventing and / or treating diseases caused by RSV infection, detecting the existence or content of the RSV antibody in a sample, diagnosing RSV infection and diagnosing the diseases caused by RSV infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to RSV pre-fusion F protein mutants and applications thereof. Background Art

[0002] Respiratory syncytial virus (RSV) was first discovered in 1956, and researchers have been trying to develop attenuated live virus vaccines or inactivated whole virus vaccine methods. However, formalin-inactivated vaccines can cause exacerbation of respiratory disease (ERD) during natural infection. Even today, the serious consequences of this incident remain the most important consideration when developing new vaccine methods and immunization strategies. With the in-depth understanding of RSV pathogenicity and vaccine-induced ERD effects, as well as continuous attempts and experience summarization in the development of monoclonal antibody drugs, attenuated live vaccines, nucleic acid vaccines, adenovirus vaccines and subunit vaccines, the successful development of RSV vaccines will usher in the dawn.

[0003] The pre-fusion state of the respiratory syncytial virus (RSV) fusion (F) glycoprotein is the main neutralizing target of antibodies in human serum, but its metastability has hindered the characterization of the pre-fusion structure. The co-crystal structure of one of the antibodies, D25, in complex with the F glycoprotein has brought hope for vaccine design. Focus on the antigenic site The introduction of the subunit vaccine DS-Cav vaccine design that fixes the RSV fusion (F) glycoprotein in a pre-fusion state provides better protection than previous vaccines. Although the prior art discloses the use of disulfide bond mutations, cavity filling mutations and electrostatic mutations to transform RSV-F proteins, the stability and / or immunogenicity of the resulting mutants still need to be improved. Therefore, it is necessary to develop a stable and / or immunogenic RSV pre-fusion F protein. Summary of the invention

[0004] The purpose of the first aspect of the present invention is to provide RSV pre-fusion F protein mutants.

[0005] The second aspect of the present invention aims to provide a composite.

[0006] The third aspect of the present invention aims to provide a fusion protein.

[0007] The fourth aspect of the present invention aims to provide a recombinant protein.

[0008] The purpose of the fifth aspect of the present invention is to provide biological materials related to the mutant of the first aspect, the fusion protein of the third aspect, and the recombinant protein of the fourth aspect of the present invention.

[0009] The sixth aspect of the present invention aims to provide a conjugate.

[0010] The purpose of the seventh aspect of the present invention is to provide applications of the mutant of the first aspect, the complex of the second aspect, the fusion protein of the third aspect, the recombinant protein of the fourth aspect, the biomaterial of the fifth aspect, and the conjugate of the sixth aspect.

[0011] The purpose of the eighth aspect of the present invention is to provide a kit.

[0012] The ninth aspect of the present invention aims to provide a medicine.

[0013] The tenth aspect of the present invention aims to provide a vaccine.

[0014] The purpose of the eleventh aspect of the present invention is to provide a method for preparing the mutant of the first aspect, the fusion protein of the third aspect, and the recombinant protein of the fourth aspect of the present invention.

[0015] The object of the twelfth aspect of the present invention is to provide a method for preventing and treating RSV infection or diseases caused by RSV infection.

[0016] The thirteenth aspect of the present invention aims to provide a method.

[0017] In order to achieve the above object, the technical solution adopted by the present invention is:

[0018] The first aspect of the present invention provides a respiratory syncytial virus (RSV) pre-fusion F protein mutant,

[0019] Compared with SEQ ID NO: 3, the RSV prefusion F protein mutant comprises at least one mutation among (1) to (3) (the obtained mutant is referred to as respiratory syncytial virus (RSV) subtype A prefusion F protein mutant):

[0020] (1) Disulfide bond mutation; (2) Cavity filling mutation; (3) Electrostatic mutation;

[0021] The disulfide bond mutations include any one of a1)-a38): a1) N105C and A147C; a2) G151C and V300C; a3) L160C and E163C; a4) G162C and N165C; a5) A170C and A177C; a6) S180C and S186C; a7) D392C and 491C; a8) E487C and A490C; a9) S491C and Q494C; a10) S62C and I199C; a11) V482C and S502C; a12) E487C, A490C, G242C and M289C; a13) E487C, A490C, T5 4C and V154C; a14) E487C, A490C, T54C and G151C; a15) E487C, A490C, S155C and S290C; a16) E487C, A490C, S55C and L188C; a17) E487C, A490C, S62C and I199C; a18) E487C, A490C, T103C and I148C; a19) E487C, A490C, R106C and V144C; a20) E487C, A490C, L138C and T337C; a21) E487C, A490C, G139C and Q354C; a22) E 487C, A490C, L142C and N371C; a23) E487C, A490C, G145C and Q370C; a24) E487C, A490C, G151C and Q302C; a25) E487C, A490C, L160C and E163C; a26) E487C and A490C, G162C and N165C; a27) E487C, A490C, A170C and A177C; a28) E487C, A490C, S180C and S186C; a29) E487C, A490C, N105C and A147C; a30) E487C, A490C, G151C and V302C; a31) S180C, S186C, S403C and T420C; a32) S180C, S186C, D392C and S491C; a33) S180C, S186C, Q34C and G471C; a34) S180C, S186C, T397C and P484C; a35) S180C, S186C, T397C and E487C; a36) S180C, S186C, S443C and S466C; a37) S180C, S186C, S491C and Q494C; a38) S180C, S186C, T482C and S502C;

[0022] The cavity filling mutation comprises any one of b1)-b40): b1) W52I; b2) L95; b3) M97L; b4) F137Y; b5) L138G; b6) F140W; b7) G144I; b8) A153F; b9) V157I; b10) V164F; b11) V192I; b12) V207L; b13) M251L; b14) V296I; b15) Q301I; b16) V308I; b17) W314F; b18) P320L; b19) L334I; b20) I395V; b21) S414I; b22) L456I; b23) V469I; b24 )I475V; b25)L481G; b26)F505W; b27)I506L; b28)L138G and V192I; b29)L138G and V207L; b30)L138G and V296I; b31)L138G and L334I; b32)L138G and F505W; b33)L 138G and F140W; b34) V207L ​​and V192I; b35) V207L ​​and V296I; b36) V207L ​​and L334I; b37) V207L ​​and F505W; b38) V296I and V192I; b39) V296I and L334I; b40) V296I and F505W;

[0023] The electrostatic mutation comprises any one of the mutations in c1)-c18): c1) E92M; c2) E92Y; c3) E92F; c4) E92W; c5) F137Y; c6) F137A; c7) N228L; c8) N228K; c9) K394M; c10) K394Y; c11) K394F; c12) D401L; c13) D401K; c14) Q501M; c15) Q501R; c16) F137Y and Q501R; c17) F137Y and N228K; c18) N228K and Q501R.

[0024] In some embodiments, the RSV pre-fusion F protein mutant comprises a disulfide bond mutation compared to SEQ ID NO:3.

[0025] In some embodiments, the RSV pre-fusion F protein mutant comprises any one of the mutations a1)-a38) compared to SEQ ID NO: 3; and further comprises the mutation described in a28).

[0026] In some embodiments, the RSV pre-fusion F protein mutant comprises a cavity-filling mutation compared to SEQ ID NO:3.

[0027] In some embodiments, the RSV pre-fusion F protein mutant comprises any one of the mutations b1)-b40) compared to SEQ ID NO:3.

[0028] In some embodiments, the RSV pre-fusion F protein mutant comprises an electrostatic mutation compared to SEQ ID NO:3.

[0029] In some embodiments, the RSV pre-fusion F protein mutant comprises any one of the mutations c1)-c18) compared to SEQ ID NO: 3; and further comprises any one of the mutations c17)-c18).

[0030] In some embodiments, the RSV pre-fusion F protein mutant comprises a disulfide bond mutation and an electrostatic mutation compared to SEQ ID NO:3.

[0031] In some embodiments, the RSV prefusion F protein mutant comprises any one of the mutations d1)-d21) compared to SEQ ID NO: 3: d1) E487C, A490C and Q501R; d2) S180C, S186C and Q501R; d3) S180C, S186C, E487C, A490C and Q501R; d4) S55C, L188C, E487C, A490C and Q501R; d5) S155C, S290C and Q501R; d6) S55C, L188C and Q501R; d7) S155C, S290C, E487C, A490C and Q501R; d8) E487C, A490C and N228K; d9) S180C, S186C and N228K; d10) S180C, S186C, E487C, A490C and N228K; d11) S55C, L188C, E487C, A490C and N22 8K; d12) S155C, S290C and N228K; d13) S55C, L188C and N228K; d14) S155C, S290C, E487C, A490C and N228K; d15) E487C, A490C, N228K and Q501R; d16) S180C, S186C, N228K and Q501R; d17) S55C, L188C, N228K and Q501R; d18) S155C, S290C, E487C, A490C, N228K and Q501R; d19) S180C, S186C, E487C, A490C, N228K and Q501R; d20) S55C, L188C, E487C, A490C, N228K and Q501R; d21) S155C, S290C, N228K and Q501R.

[0032] In some embodiments, the RSV pre-fusion F protein mutant comprises a disulfide bond mutation and a cavity filling mutation compared to SEQ ID NO:3.

[0033] In some embodiments, the RSV prefusion F protein mutant comprises any one of the mutations e1)-e28) compared to SEQ ID NO: 3: e1) E487C, A490C and V207L; e2) S180C, S186C and V207L; e3) S180C, S186C, E487C, A490C and V207L; e4) S55C, L188C, E487C, A490C and V207L; e5) S155C, S290C and V207L; e6) S55C, L188C and V207L; e7) S155C, S290C, E487C, A490C and V207L; e8) E487C, A490C and V207L; 0C and V296I; e9) S180C, S186C and V296I; e10) S180C, S186C, E487C, A490C and V296I; e11) S55C, L188C, E487C, A490C and V296I; e12) S155C, S290C and V296I; e13) S55C, L188C and V296I; e14) S155C, S290C, E487C, A490C and V296I; e15) S55C, L188C, E487C, A490C, V207L ​​and L334I; e16) S 155C, S290C, V207L ​​and L334I; e17) S55C, L188C, V207L ​​and L334I; e18) S155C, S290C, E487C, A490C, V207L ​​and L334I; e19) ​​E487C, A490C, V296I and L334I; e20) S180C, S186C, V296I and L334I; e21) S180C, S186C, E487C, A490C, V296I and L334I; e22) S55C, L188C, E487C, A490C, V296I and L334I; e23) S155C, S290C, V296I and L334I; e24) S55C, L188C, V296I and L334I; e25) S155C, S290C, E487C, A490C, V296I and L334I; e26) E487C, A490C, V207L ​​and L334I; e27) S180C, S186C, V207L ​​and L334I; e28) S180C, S186C, E487C, A490C, V207L ​​and L334I; further comprising any one of the mutations in e4) and e11).

[0034] In some embodiments, the RSV pre-fusion F protein mutant comprises a disulfide bond mutation, a cavity filling mutation, and an electrostatic mutation compared to SEQ ID NO:3.

[0035] In some embodiments, the RSV pre-fusion F protein mutant is identical to SEQ ID NO:3, comprising any one of the mutations f1)-f84): f1) E487C, A490C, V207L ​​and Q501R; f2) S180C, S186C, V207L ​​and Q501R; f3) S180C, S186C, E487C, A490C, V207L ​​and Q501R; f4) S55C, L188C, E487C, A490C, V207L ​​and Q501R; f5) S155C, S290C, V207L ​​and Q501R; f6) S180C, S186C, V296I and Q501R; f7) S180C, S186C, E487C, A490C, V296I and Q501R; f8) S55C, L188C, E487C, A490C, V296I and Q501R; f9) S155C, S290C, V296I and Q501R; f10) S55C, L188C, V296I and Q501R; f11) S155C, S290C, E487C, A490C, V296I and Q501R; f12) E487C, A490C, V207L, L334I and Q501R; f13) S180C, S186C, V207L, L334I and Q501R; f14) S180C, S186C, E487C, A49 0C, V207L, L334I and Q501R; f15) S55C, L188C, E487C, A490C, V207L, L334I and Q501R; f16) S155C, S290C, V207L, L334I and Q501R; f17) S55C, L188C, V207L, L334I and Q501R; f18) S155C, S290C, E487C, A490C, V207L, L334I and Q501R; f19) E487C, A490C, V296I, L334I and Q501R; f20) S180C, S186C, V296I, L3 34I and Q501R; f21) S180C, S186C, E487C, A490C, V296I, L334I and Q501R; f22) S155C, S290C, E487C, A490C, V296I, L334I and Q501R; f23) E487C, A490C, V207L ​​and N228K; f24) S180C, S186C, V207L ​​and N228K; f25) S180C, S186C, E487C, A490C, V207L ​​and N228K; f26) S55C, L188C, E487C, A490C, V207L ​​and N228K;f27) S155C, S290C, V207L ​​and N228K; f28) S55C, L188C, V207L ​​and N228K; f29) S155C, S290C, E487C, A490C, V207L ​​and N228K; f30) E487C, A490C, V296I and N228K; f31) S180C, S186C, V296I and N228K; f32) S180C, S186C, E487C, A490C, V2 96I and N228K; f33) S55C, L188C, E487C, A490C, V296I and N228K; f34) S155C, S290C, V296I and N228K; f35) S55C, L188C, V296I and N228K; f36) S155C, S290C, E487C, A490C, V296I and N228K; f37) S155C, S290C, V207L, L334I and N228K; f38) S55C, L188C, V207L, L334I and N228K; f39) S155C, S290C, E487C, A490C, V207L, L334I and N228K; f40) E487C, A490C, V296I, L334I and N228K; f41) S180C, S186C, V296I, L334I and N228K; f42) S180C, S186C, E487C, A490C, V296I, L334 I and N228K; f43) S55C, L188C, E487C, A490C, V296I, L334I and N228K; f44) S155C, S290C, V296I, L334I and N228K; f45) S55C, L188C, V296I, L334I and N228K; f46) S155C, S290C, E487C, A490C, V296I, L334I and N228K; f47) E487C, A490C;

[0036] , V207L, N228K and Q501R; f48) S180C, S186C, V207L, N228K and Q501R; f49) S180C, S186C, E487C, A490C, V207L, N228K and Q501R; f50) S55C, L188C, E487C, A490C, V207L, N228K and Q501R; f51) S155C, S290C, V207L, N228K and Q501R; f52) S55C, L188C, V207L, N228K and Q501R; f53) S180C, S186C, E487C, A490C, V296 I, N228K and Q501R; f54) S55C, L188C, E487C, A490C, V296I, N228K and Q501R; f55) S155C, S290C, V296I, N228K and Q501R; f56) S55C, L188C, V296I, N228K and Q501R; f57) S155C, S290C, E487C, A490C, V296I, N228K and Q501R; f58) E487C, A490C, V207L, L334I, N228K and Q501R; f59) S180C, S186C, V207L, L334I, N228 K and Q501R; f60) S180C, S186C, E487C, A490C, V207L, L334I, N228K and Q501R; f61) S55C, L188C, E487C, A490C, V207L, L334I, N228K and Q501R; f62) S155C, S290C, V207L, L334I, N228K and Q501R; f63) S55C, L188C, V207L, L334I, N228K and Q501R; f64) S155C, S290C, E487C, A490C, V207L, L334I, N228K and Q501R; f 65) E487C, A490C, V296I, L334I, N228K and Q501R; f66) S180C, S186C, V296I, L334I, N228K and Q501R; f67) S180C, S186C, E487C, A490C, V296I, L334I, N228K and Q501R; f68) S55C, L188C, E487C, A490C, V296I, L334I, N228K and Q501R; f69) E487C, A490C, V207L, L334I, and N228K; f70) S55C, L188C, V207L ​​and Q501R;f71) S155C, S290C, E487C, A490C, V207L ​​and Q501R; f72) E487C, A490C, V296I and Q501R; f73) S55C, L188C, E487C, A490C, V296I, L334I and Q501R; f74) S155C, S290C, V296I, L334I and Q501R; f75) S 55C, L188C, V296I, L334I and Q501R; f76) S180C, S186C, V207L, L334I and N228K; f77) S180C, S186C, E487C, A490C, V207L, L334I and N228K; f78) S55C, L188C, E487C, A490C, V207L, L334I and N228K ; f79) S155C, S290C, E487C, A490C, V207L, N228K and Q501R; f80) E487C, A490C, V296I, N228K and Q501R; f81) S180C, S186C, V296I, N228K and Q501R; f82) S155C, S290C, V296I, L334I, N228K and Q50 1R; f83) S55C, L188C, V296I, L334I, N228K and Q501R; f84) S155C, S290C, E487C, A490C, V296I, L334I, N228K and Q501R; further comprising any one of f7), f14), f23), f25), f26), f47), f49), f54), f60), and f78). ;

[0037] The second aspect of the present invention provides a complex comprising the RSV pre-fusion F protein mutant of the first aspect of the present invention;

[0038] and vector components capable of displaying the mutants.

[0039] In some embodiments, the carrier component comprises at least one of a nanomaterial, a bacterial outer membrane vesicle (OMVs), a polymerizing base, and a virus-like particle (VLP).

[0040] In some embodiments, the nanomaterial comprises at least one of lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers, and biomimetic nanoparticles.

[0041] In some embodiments, the VLP is assembled from proteins obtained from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

[0042] The third aspect of the present invention provides a fusion protein comprising the RSV pre-fusion F protein mutant of the first aspect of the present invention;

[0043] and a trimerization base.

[0044] In some embodiments, the amino acid sequence of the trimerization base is as shown in SEQ ID NO:4.

[0045] In some embodiments, the fusion protein comprises the RSV pre-fusion F protein mutant of the first aspect of the present invention from N-terminus to C-terminus, and a trimerization base.

[0046] The fourth aspect of the present invention provides a recombinant protein comprising the fusion protein of the third aspect of the present invention;

[0047] and optionally a tag sequence to facilitate expression and / or purification.

[0048] In some embodiments, the tag sequence is selected from at least one of His tag, FLAG, Strep-Tag II, Polyarg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, and NusA.

[0049] In some embodiments, the recombinant protein comprises the fusion protein of the third aspect of the present invention from the N-terminus to the C-terminus, and optionally a tag sequence that facilitates expression and / or purification.

[0050] A fifth aspect of the present invention provides a biological material related to the RSV pre-fusion F protein mutant of the first aspect of the present invention, the fusion protein of the third aspect, or the recombinant protein of the fourth aspect, wherein the biological material comprises any one of n1)-n9):

[0051] n1) a nucleic acid molecule encoding the RSV pre-fusion F protein mutant of the first aspect, the fusion protein of the third aspect, or the recombinant protein of the fourth aspect of the present invention;

[0052] n2) an expression cassette comprising the nucleic acid molecule described in n1);

[0053] n3) a vector comprising the nucleic acid molecule described in n1);

[0054] n4) a vector comprising the expression cassette described in n2);

[0055] n5) a cell comprising the nucleic acid molecule described in n1);

[0056] n6) a cell comprising the expression cassette described in n2);

[0057] n7) a cell comprising the vector described in n3);

[0058] n8) a cell comprising the vector described in n4);

[0059] n9) A cell comprising the RSV pre-fusion F protein mutant of the first aspect, the fusion protein of the third aspect, or the recombinant protein of the fourth aspect of the invention.

[0060] In some embodiments, the cell of any one of n5)-n9) does not contain propagation material.

[0061] In some embodiments, any of the vectors n3)-n4) include a prokaryotic expression vector and a eukaryotic expression vector.

[0062] In some embodiments, the eukaryotic expression vector includes a yeast expression vector, a mammalian expression vector, an insect expression vector, and the like.

[0063] In some embodiments, the cell described in any one of n5)-n9) is selected from a prokaryotic cell and a eukaryotic cell.

[0064] In some embodiments, the prokaryotic cells include bacterial cells, Escherichia coli, and Streptomyces.

[0065] In some embodiments, the eukaryotic cells include yeast cells, mammalian cells, insect cells, etc.

[0066] In some embodiments, the mammal is selected from humans, monkeys, mice, rats, hamsters, goats, sheep, cows, pigs, dogs, and cats.

[0067] The sixth aspect of the present invention provides a conjugate comprising the RSV pre-fusion F protein mutant of the first aspect of the present invention, the fusion protein of the third aspect, or the recombinant protein of the fourth aspect;

[0068] and a coupling moiety, wherein the coupling moiety is a detectable label.

[0069] In some embodiments, the detectable label comprises at least one of a metal particle, a fluorescent label, a luminescent label, an electron dense label, a chemiluminescent label, a radioactive label, and an enzyme label.

[0070] In some embodiments, the detectable label comprises at least one of a radioisotope, a fluorophore, a luciferase, a horseradish peroxidase, an alkaline phosphatase, a β-galactosidase, a glucoamylase, a lysozyme, a carbohydrate oxidase, a glucose-6-phosphate dehydrogenase, a rhodamine, a fluorescein, a glucose oxidase, a galactose oxidase, an acridinium ester, an acridinium sulfonamide, a luminol, and an isoluminol.

[0071] In some embodiments, the radioactive isotope is selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, P-32, H-3, S-35, Lu-177 and Re-188.

[0072] The seventh aspect of the present invention provides the use of the RSV pre-fusion F protein mutant of the first aspect of the present invention, the complex of the second aspect, the fusion protein of the third aspect, the recombinant protein of the fourth aspect, or the biomaterial of the fifth aspect in any one of m1)-m2);

[0073] m1) preparing RSV antibodies;

[0074] m2) preparing medicines;

[0075] The drug is used for 11) or 12):

[0076] l1) Prevention of RSV infection;

[0077] 12) Prevent and / or treat diseases caused by RSV infection.

[0078] Use of the RSV pre-fusion F protein mutant of the first aspect, the fusion protein of the third aspect, the recombinant protein of the fourth aspect, the biomaterial of the fifth aspect, or the conjugate of the sixth aspect of the present invention in preparing a kit;

[0079] The kit is used for any one of o1)-o3):

[0080] o1) detecting the presence or amount of RSV antibodies in a sample;

[0081] o2) Diagnosis of RSV infection;

[0082] o3) Diagnosis of diseases caused by RSV infection.

[0083] In some embodiments, the RSV in o1)-o3) comprises RSV A subtype.

[0084] In some embodiments, the disease caused by the RSV infection comprises at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease and cardiopulmonary complications.

[0085] The eighth aspect of the present invention provides a kit comprising: the RSV pre-fusion F protein mutant of the first aspect of the present invention, the fusion protein of the third aspect, the recombinant protein of the fourth aspect, or the conjugate of the sixth aspect.

[0086] In some embodiments, the kit is used for any one of o1)-o3):

[0087] o1) detecting the presence or amount of RSV antibodies in a sample;

[0088] o2) Diagnosis of RSV infection;

[0089] o3) Diagnosis of diseases caused by RSV infection.

[0090] In some embodiments, the RSV in o1)-o3) comprises RSV A subtype.

[0091] In some embodiments, the disease caused by the RSV infection comprises at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease and cardiopulmonary complications.

[0092] The ninth aspect of the present invention provides a drug comprising: the RSV pre-fusion F protein mutant of the first aspect of the present invention, the complex of the second aspect, the fusion protein of the third aspect, the recombinant protein of the fourth aspect, or the biological material of the fifth aspect.

[0093] In some embodiments, the drug further comprises: a pharmaceutically acceptable carrier.

[0094] In some embodiments, the medicine further comprises: other active ingredients for preventing and / or treating RSV infection, or diseases caused by RSV infection.

[0095] In some embodiments, the medicament is used for 11) or 12):

[0096] l1) prevention and / or treatment of RSV infection;

[0097] 12) Prevent and / or treat diseases caused by RSV infection.

[0098] In some embodiments, the RSV comprises RSV A subtype.

[0099] In some embodiments, the disease caused by the RSV infection comprises at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease and cardiopulmonary complications.

[0100] The tenth aspect of the present invention provides a vaccine comprising: the RSV pre-fusion F protein mutant of the first aspect of the present invention, the complex of the second aspect, the fusion protein of the third aspect, the recombinant protein of the fourth aspect, or the biological material of the fifth aspect;

[0101] and adjuvants.

[0102] In some embodiments, the vaccine is used for 11) or 12):

[0103] l1) prevention and / or treatment of RSV infection;

[0104] 12) Prevent and / or treat diseases caused by RSV infection.

[0105] In some embodiments, the RSV in 11)-12) comprises RSV A subtype.

[0106] In some embodiments, the disease caused by the RSV infection comprises at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease and cardiopulmonary complications.

[0107] The eleventh aspect of the present invention provides a method for preparing the RSV pre-fusion F protein mutant of the first aspect of the present invention, the fusion protein of the third aspect, and the recombinant protein of the fourth aspect, which are obtained by culturing the cells in the fifth aspect of the present invention.

[0108] The twelfth aspect of the present invention provides a method for preventing and treating RSV infection or a disease caused by RSV infection, comprising administering an effective amount of the medicament of the ninth aspect of the present invention or the vaccine of the tenth aspect of the present invention to a subject.

[0109] In some embodiments, the RSV comprises RSV A subtype.

[0110] In some embodiments, the disease caused by the RSV infection comprises at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease and cardiopulmonary complications.

[0111] In some embodiments, the subject can include a mammal, such as a human or a non-human mammal.

[0112] In some embodiments, the non-human mammals may include, but are not limited to, non-human primates (e.g., monkeys, gorillas), mice, rats, hamsters, gerbils, cats, dogs, guinea pigs, rabbits, horses, sheep, cows, pigs, etc.

[0113] The thirteenth aspect of the present invention provides a method comprising the steps of using the kit of the eighth aspect of the present invention;

[0114] The method is used for any one of o1)-o3):

[0115] o1) detecting the presence or amount of RSV antibodies in a sample;

[0116] o2) Diagnosis of RSV infection;

[0117] o3) Diagnosis of diseases caused by RSV infection.

[0118] In some embodiments, the RSV in o1)-o3) comprises RSV A subtype.

[0119] In some embodiments, the disease caused by the RSV infection comprises at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease and cardiopulmonary complications.

[0120] The beneficial effects of the present invention are:

[0121] The present invention provides a respiratory syncytial virus (RSV) prefusion F protein mutant, which has good stability, high expression level and / or immunogenicity, and can be used to prepare RSV antibodies, prevent RSV infection, prevent and / or treat diseases caused by RSV infection, detect the presence or content of RSV antibodies in a sample, diagnose RSV infection, and diagnose diseases caused by RSV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 The neutralization titer NT50 of RSV by the sera of mice immunized with the mutants is shown. DETAILED DESCRIPTION

[0123] The present invention is further described in detail below through specific examples.

[0124] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0125] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials and reagents used in the examples are commercially available unless otherwise specified. The manufacturers of the reagents used are indicated, and similar products from other manufacturers are substitutes.

[0126] Example 1

[0127] 1. Construction of plasmid expressing RSV prefusion F protein mutant (RSV A subtype prefusion F protein mutant)

[0128]

[0129] The PCR reaction system for introducing point mutations is shown in Table 1, some of the primer sequences used are shown in Table 2, and the homologous recombination reaction system and conditions are shown in Table 3.

[0130] The plasmid construction, PCR reaction system and homologous recombination process of RSV prefusion F protein mutants DS-CAV, hd1b-1, hd3b-1, hd3c-2, hd4b-1, hd1c-3, hf3a-1, hf3c-1, hd4-2, he3a-3, hf4b-1, he4c-1, he4-1, hg1b-1, he3b-3, he1c-1 and hf3-3 are taken as examples:

[0131] (1) Construction of DS-CAV: ① In the first round, WT-F plasmid was used as a template, and the short PCR fragment was amplified using primers (S155C-F; S290C-R), and the long fragment was amplified using primers (S155C-R; S290C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. ② In the second round of PCR, the plasmid constructed in ① was used as a template, and the short PCR fragment was amplified using primers (S190F-F; V207L-R), and the long fragment was amplified using primers (S190F-R; V207L-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. (2) Construction of hd1b-1: ① In the first round, WT-F plasmid was used as a template, and the short PCR fragment was amplified using primers (V207L-F; E487C, A490C-R), and the long fragment was amplified using primers (V207L-R; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. ② In the second round of PCR, the plasmid constructed in ① was used as a template, and the short PCR fragment was amplified using primers (N228K-F; E487C, A490C-R), and the long fragment was amplified using primers (N228K-R; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. (3) Construction of hd1c-3: Using hd1b-1 plasmid as template, the short PCR fragment was amplified using primers (N228K-F; Q501R-R), and the long fragment was amplified using primers (N228K-R; Q501R-F). The primer sequences are shown in Table 2. Other reagents are shown in Table 1. The PCR products were subjected to homologous recombination using the reaction system in Table 3. (4) Construction of hd3b-1: Using hd1b-1 plasmid as template, the short PCR fragment was amplified using primers (S180C, S186C-F; E487C, A490C-R), and the long fragment was amplified using primers (S180C, S186C; E487C, A490C-F). The primer sequences are shown in Table 2. Other reagents are shown in Table 1. The PCR products were subjected to homologous recombination using the reaction system in Table 3. (5) Construction of hd3c-2: Using hd1c-3 plasmid as template, the short PCR fragment was amplified using primers (S180C, S186C-F; E487C, A490C-R), and the long fragment was amplified using primers (S180C, S186C; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were subjected to homologous recombination using the reaction system in Table 3.(6) Construction of hd4-2: ① In the first round, WT-F plasmid was used as a template, and the short PCR fragment was amplified using primers (S55C-F; L188C-R), and the long fragment was amplified using primers (S55C-R; L188C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. ② In the second round of PCR, the plasmid constructed in ① was used as a template, and the short PCR fragment was amplified using primers (V207L-RF; E487C, A490C-R), and the long fragment was amplified using primers (V207L-RR; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. (7) Construction of hd4b-1: Using hd4-2 plasmid as template, the short PCR fragment was amplified using primers (N228K-F; E487C, A490C-R), and the long fragment was amplified using primers (N228K-R; E487C, A490C-F). The primer sequences are shown in Table 2. Other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. (8) Construction of he1c-1: ① In the first round, using WT-F plasmid as template, the short PCR fragment was amplified using primers (V296I-F; E487C, A490C-R), and the long fragment was amplified using primers (V296I-R; E487C, A490C-F). The primer sequences are shown in Table 2. Other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. ② The second round of PCR used the plasmid constructed in ① as a template, and the short PCR fragment was amplified using primers (N228K-F; Q501R-R), and the long fragment was amplified using primers (N228K-R; Q501R-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. (9) Construction of he3a-3: Using the plasmid constructed in (8) ① as a template, the short PCR fragment was amplified using primers (S180C, S186C-F; Q501R-R), and the long fragment was amplified using primers (S180C, S186C-R; Q501R-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. (10) he3b-3 construction: Using (8)① plasmid as template, the short PCR fragment was amplified using primers (S180C, S186C-F; N228K-R), and the long fragment was amplified using primers (S180C, S186C-R; QN228K-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were subjected to homologous recombination using the reaction system in Table 3.(11) he4-1 construction: using (8)① plasmid as template, PCR short fragment was amplified using primers (S55C-F; L188C-R), long fragment was amplified using primers (S55C-R; L188C-F), primer sequences are shown in Table 2, other reagents are shown in Table 1, and PCR products were homologously recombined using the reaction system in Table 3. (12) he4c-1 construction: using he4-1 plasmid as template, PCR short fragment primers (N228K-F; Q501R-R), long fragment primers (N228K-R; Q501R-F), primer sequences are shown in Table 2, other reagents are shown in Table 1, and PCR products were homologously recombined using the reaction system in Table 3. (13) Construction of hf3-3: ① In the first round, WT-F plasmid was used as template, and the short PCR fragment was amplified using primers (V207L-F; L334I-R), and the long fragment was amplified using primers (V207L-R; L334I-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were subjected to homologous recombination using the reaction system in Table 3. ② In the second round of PCR, the plasmid constructed in ① was used as template, and the short PCR fragment was amplified using primers (S180C, S186C-F; E487C, A490C-R), and the long fragment was amplified using primers (S180C, S186C-R; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were subjected to homologous recombination using the reaction system in Table 3. (14) Construction of hf3c-1: Using the hf3-3 plasmid as a template, the short PCR fragment was amplified using primers (N228K-F; Q501R-R), and the long fragment was amplified using primers (N228K-R; Q501R-F). The primer sequences are shown in Table 2. Other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. (15) Construction of hf3a-1: Using the hf3-3 plasmid as a template, the short PCR fragment was amplified using primers (V207L-F; Q501R-R), and the long fragment was amplified using primers (V207L-R; Q501R-F). The primer sequences are shown in Table 2. Other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. (16) Construction of hf4b-1: ① In the first round, (13)① plasmid was used as template, and the short PCR fragment was amplified using primers (N228K-F; E487C, A490C-R), and the long fragment was amplified using primers (N228K-R; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were subjected to homologous recombination using the reaction system in Table 3.② The second round of PCR used the plasmid constructed in ① as a template, and the short PCR fragment was amplified using primers (S55C-F; L188C-R), and the long fragment was amplified using primers (S55C-R; L188C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. (17) hg1b-1 construction: ① The first round used the WT-F plasmid as a template, and the short PCR fragment was amplified using primers (V207L-RF; E487C, A490C-R), and the long fragment was amplified using primers (V207L-RR; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3. ② The second round of PCR used the plasmid constructed in ① as a template, the PCR short fragment was amplified using primers (V207L-F; V296I-R), and the long fragment was amplified using primers (V207L-R; V296I-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were homologously recombined using the reaction system in Table 3.

[0132] Table 1 PCR reaction system

[0133] 2xphanta max buffer 25μL dNTP 1μL F 2μL R 2μL DNA polymerase 1μL Plasmid template 1μL(50ng) <![CDATA[H2O]]> 18μL Total 50μL

[0134] Table 2 Primer sequences

[0135]

[0136]

[0137] Table 3 Homologous recombination reaction system and conditions

[0138]

[0139] The constructed plasmids expressing RSV prefusion F protein mutants were transformed into Escherichia coli DH5α competent cells, spread on solid LB culture dishes (final Amp concentration of 100 μg / mL), cultured overnight, picked bacteria for amplification in liquid LB (final Amp concentration of 100 μg / mL), and sent for sequencing identification. The plasmid expressing RSV prefusion F protein mutant (the amino acid sequence of the protein expressed by the plasmid expressing RSV prefusion F protein mutant compared to the mutation site in SEQ ID NO: 2 is shown in Table 4) was successfully constructed.

[0140] Table 4 Mutation sites corresponding to each mutant

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] 2. Testing the stability of mutants

[0147] The stability of mutants was tested as follows:

[0148] 1) Use 96-well plate with 1x10 per well 4 / 100 μL 293t cells were plated, and the plasmid of RSV pre-fusion F protein mutant was transfected with PEI on the second day (100 ng / well). On the third day, 100 μL of culture medium containing 10% FBS was added and cultured for another 4 days;

[0149] 2) The cell culture supernatant with the same density obtained in 1) was treated as follows: placed at 4°C for 1 week (4°C-1w), placed at 50°C for 1h (50°C-1h), and / or placed at 60°C for 1h (60°C-1h), and then subjected to sandwich ELISA detection.

[0150] The sandwich ELISA detection method is as follows: 1) One day in advance, use 100 ng / well of D25 (Biodragon / Bo Ao Long, BD-VA1156) or AM14 (Biodragon / Bo Ao Long, BD-VA1155) antibody to coat overnight; 2) The treated or untreated cell culture supernatant was diluted 5-125 times or not diluted (the specific dilution is shown in Table 5-10), and the treated or untreated cell culture supernatant was added to the 96-well plate coated with antibody and washed 3 times with PBST at 100 μL / well and incubated overnight; 3) Wash 3 times with PBST, add 50 ng / well of anti-his antibody (Proteintech / Sanying Biological, 66005-1-G-1000UL), and leave at room temperature for 1.5 hours; 4) Wash 3 times with PBST, add 100 ng / well of mouse secondary antibody with HRP (Jackson immuno research, 115-035-146), room temperature for 1 hour; 5) washed 3 times with PBST, treated with ELISA developer for 15 minutes, and read the OD450 value immediately after termination with stop solution.

[0151] The mutant stability results are shown in Tables 5, 6, 7, 8, 9, and 10 (since the stability analysis was performed in different batches, some mutant results were repeated): the above mutants had good stability, especially D5, D17, G3, hb3-4, hb4-1, hb7-2, hc2-2, hc7-3, hd4-2, hd5-1, hd7-1, he4-1, he7-1, hf4-3, hf7-1, hg3-1, hg4-2, hg6-1, hg7-1, hd3a-2, hd4a-2, he4a-3, hf3a-1, hf4a-3, hf7a-2, hg7a-2, hd1b- 1. hd2b-1, hd3b-1, hd4b-1, hd5b-1, hd6b(2)-1, hd7b-2, he1b-1, he2b-1, he3b-3, he4b-2, he5 b-2, he6b-1, he7b-1, hf1b-1, hf5b-1, hf6b-2, hf7b-1, hg1b-1, hg2b-3, hg3b-1, hg4b-1, hg5 b-1, hg6b-1, hg7b-1, hd1c-3, hd2c-1, hd3c-2, hd4c-1, hd5(2)-1, hd6c-1, he3c(3)-2, he4c-1 , he5c-1, he6c-1, he7c-3, hf2c-2, hf3c-1, hf4c-1, hf5c-1, hf6c-2, hf7c-1, hg2c-1, hg3c-2, hc3-4, hc4-1(2), hc5-1, hf3-3, hd7a(2)-1, hf4b-1, and he1c-1 were superior to or comparable to DS-CAV (at least under 50°C-1h treatment).

[0152] Table 5 Disulfide bond single mutation results (5-fold dilution)

[0153]

[0154] Note: “D25” means detection using D25 antibody.

[0155] Table 6 Disulfide bond double mutation results (5-fold dilution)

[0156]

[0157]

[0158] Note: “D25” means detection using D25 antibody.

[0159] Table 7 Electrostatic single mutation results (5-fold dilution)

[0160]

[0161]

[0162] Note: “D25” means detection using D25 antibody.

[0163] Table 8 Cavity single mutation results (undiluted)

[0164]

[0165]

[0166] Note: “D25” means detection using D25 antibody.

[0167] Table 9 Electrostatic / cavity double mutation results (5-fold dilution)

[0168]

[0169] Note: “D25” means detection using D25 antibody.

[0170] Table 10 Three types of combined mutation results (n = 3) (125-fold dilution)

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] Note: “D25” means the detection is done with D25 antibody, and “AM14” means the detection is done with AM14 antibody.

[0177] 3. Affinity determination of RSV-F and related neutralizing antibodies

[0178] From the 133 combinations of disulfide bond mutations, cavity filling mutations and electrostatic mutations (Table 10), 36 mutant combinations (Table 11) were selected for affinity testing:

[0179] 1) When the 293f cell density reaches 2x10 6 / 100μL, transfected with PEI (the amount of transfection plasmid was 2μg / mL);

[0180] 2) Collect the cell suspension, centrifuge at 1000 rpm for 10 min to remove cells, 8000 rpm for 30 min to remove cell debris, and filter with a 0.45 μm pore size filter;

[0181] 3) Using Amicon Ultra filter, 30 kDa MWCO (UFC5030), concentrate the filtrate and replace the buffer with PBS;

[0182] 4) Biacore 8K surface plasmon resonance (SPR) was used to test the affinity of the above mutants with neutralizing antibodies D25 (Biodragon / Biodragon, BD-VA1156), AM14 (Biodragon / Biodragon, BD-VA1155), AM22 (Biodragon / Biodragon, BD-VA1358), 101F (AntibodySystem, AntibodySystem_RVV02816), and MOTE (Prosci, 10-089) (refer to the instructions for the detection method).

[0183] The results are shown in Table 11: The above mutants have good affinity with neutralizing antibodies against RSV-F.

[0184] Table 11 Antigen epitope affinity results

[0185]

[0186]

[0187] 4. Test expression

[0188] Based on the thermal stability and affinity test data, 16 mutant proteins (Table 12) were screened for expression test

[0189] 1) Detect the expression of some mutants as follows: Use 1x10 4 / 100μL 293t cells were plated, and the plasmid (100ng / well) was transfected with PEI on the second day. On the third day, 100μL of culture medium containing 10% FBS was added, and cultured for another 4 days. The secreted F protein in the cell culture supernatant was detected using sandwich ELISA (the method is the same as "2. Detection of the stability of mutants"). The results are shown in Table 12: The expression levels of mutants hd1b-1, hd1c-3, hd3b-1, hd3c-2, hd4-2, hd4b-1, he1c-1, he3a-3, he3b-3, he4c-1, he4-1, hf3-3, hf3c-1, hf3a-1, hf4b-1, and hg1b-1 were all higher than or equivalent to DS-CAV.

[0190] Table 12 Expression levels of some mutants (n=3)

[0191]

[0192]

[0193] Note: “D25” means detection using D25 antibody.

[0194] 5. Expression and purification of WT, DS-CAV, hd1b-1, hd3b-1, hd3c-2, hd4b-1, hd1c-3, hf3a-1, hf3c-1, hd4-2, he3a-3, hf4b-1, he4c-1, and he4-1

[0195] 1) When the 293f cell density reaches 2x10 6 / 100μL, transfected with PEI (the amount of transfection plasmid was 2μg / mL);

[0196] 2) 6 days after transfection, the cell suspension was collected and centrifuged at 1000 rpm for 10 min and 8000 rpm for 30 min to remove cells and cell debris to obtain cell supernatant;

[0197] 3) Filter the supernatant again by suction filtration to prevent clogging of the purification column;

[0198] 4) Add a nickel column to the purification column, pass the supernatant through the column three times, and then elute;

[0199] 5) Concentrate the eluted protein solution using a concentrator tube and replace the buffer with 1X PBS;

[0200] 6) Determine the protein concentration using the BCA protein assay;

[0201] 7) Take 10 μL of the protein sample after the protein concentration test, add 5x loading buffer, heat denaturation at 98°C for 10 min, and perform electrophoresis using SDS-PAGE gel;

[0202] 8) Use Coomassie blue to stain the protein and determine the protein purity.

[0203] 6. Mouse Immunization

[0204] Five Balb / c mice were immunized with each protein antigen, and 50 μL of the protein solution obtained in 3 above (protein concentration was 2 μg / 100 μL) was injected intramuscularly into each of the left and right thighs. After the first immunization, a second immunization with the same dose was performed on the 21st day. Blood was collected from the eyeballs on the 35th day, and serum was separated for subsequent neutralization experiments.

[0205] 7. Neutralization experiment

[0206] 1) Use medium containing 2% fetal bovine serum at a rate of 1 x 10 4 / 100 μL hep2 cells were plated;

[0207] 2) The mouse serum obtained in step 6 was uniformly diluted 10-fold, and then graded diluted 3-fold (the diluent was 100 TCID50 RSV virus solution) (GenBank: MW582527.1), with a total of 7 dilution gradients. After mixing, incubate at 37°C for 1 h, inverting and shaking once during the dilution;

[0208] 3) Add the diluted serum in 2) to the well plate in 1), 100 μL per well, 6 replicate wells per dilution gradient, and set up Yang Shen (400 TCID50 RSV virus solution) and Yin Shen (culture medium containing 2% fetal bovine serum) at the same time;

[0209] 4) After two days of culture, remove the supernatant PBS twice, add 4% paraformaldehyde, and seal with sealing glue;

[0210] 5) Use high-content plate scanning to count the fluorescence area of ​​each well and perform neutralization data analysis;

[0211] 6) Use the analysis function of GraphPad Prism to calculate the neutralization NT50.

[0212] The results are as follows Figure 1 As shown in the results, the above mutants have good immunogenicity (all better than WT), especially hd3b-1, hd3c-2, hd1c-3, hf3a-1, hf3c-1, hf4b-1 and he4-1 are better than DSCAV.

[0213] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A respiratory syncytial virus (RSV) prefusion F protein mutant, wherein the RSV prefusion F protein mutant comprises at least one mutation of (1)-(3) compared with SEQ ID NO: 3: (1) Disulfide bond mutation; (2) Cavity filling mutation; (3) Electrostatic mutation; The disulfide bond mutations include any one of a1)-a38): a1) N105C and A147C; a2) G151C and V300C; a3) L160C and E163C; a4) G162C and N165C; a5) A170C and A177C; a6) S180C and S186C; a7) D392C and 491C; a8) E487C and A490C; a9) S491C and Q494C; a10) S62C and I199C; a11) V482C and S502C; a12) E487C, A490C, G242C and M289C; a13) E487C, A490C, T5 4C and V154C; a14) E487C, A490C, T54C and G151C; a15) E487C, A490C, S155C and S290C; a16) E487C, A490C, S55C and L188C; a17) E487C, A490C, S62C and I199C; a18) E487C, A490C, T103C and I148C; a19) E487C, A490C, R106C and V144C; a20) E487C, A490C, L138C and T337C; a21) E487C, A490C, G139C and Q354C; a22) E 487C, A490C, L142C and N371C; a23) E487C, A490C, G145C and Q370C; a24) E487C, A490C, G151C and Q302C; a25) E487C, A490C, L160C and E163C; a26) E487C and A490C, G162C and N165C; a27) E487C, A490C, A170C and A177C; a28) E487C, A490C, S180C and S186C; a29) E487C, A490C, N105C and A147C; a30) E487C, A490C, G151C and V302C; a31) S180C, S186C, S403C and T420C; a32) S180C, S186C, D392C and S491C; a33) S180C, S186C, Q34C and G471C; a34) S180C, S186C, T397C and P484C; a35) S180C, S186C, T397C and E487C; a36) S180C, S186C, S443C and S466C; a37) S180C, S186C, S491C and Q494C; a38) S180C, S186C, T482C and S502C; The cavity filling mutation comprises any one of b1)-b40): b1) W52I; b2) L95; b3) M97L; b4) F137Y; b5) L138G; b6) F140W; b7)G144I; b8)A153F; b9)V157I; b10)V164F; b11)V192I; b12) V207L; b13)M251L; b14)V296I; b15)Q301I; b16)V308I; b17)W314F; b18)P320L; b19)L334I; b20)I395V; b21)S414I; b22)L456I; b23)V469I; b24)I475V; b25)L481G; b26)F505W; b27)I506L; b28)L138G and V192I; b29) L138G and V207L; b30) L138G and V296I; b31) L138G and L334I; b32) L138G and F505W; b33) L138G and F140W; b34) V207L ​​and V192I; b35) V207L ​​and V296I; b36) V207L ​​and L334I; b37)V207L ​​and F505W; b38) V296I and V192I; b39) V296I and L334I; b40) V296I and F505W; The electrostatic mutation comprises any one of the mutations in c1)-c18): c1) E92M; c2) E92Y; c3) E92F; c4) E92W; c5) F137Y; c6) F137A; c7) N228L; c8) N228K; c9) K394M; c10) K394Y; c11) K394F; c12) D401L; c13) D401K; c14) Q501M; c15) Q501R; c16) F137Y and Q501R; c17) F137Y and N228K; c18) N228K and Q501R.

2. The RSV prefusion F protein mutant according to claim 1, characterized in that The RSV pre-fusion F protein mutant comprises a disulfide bond mutation compared to SEQ ID NO: 3; Preferably, the RSV pre-fusion F protein mutant comprises any one of a1)-a38) mutations compared to SEQ ID NO: 3; Preferably, the RSV prefusion F protein mutant comprises a cavity-filling mutation compared to SEQ ID NO: 3; Preferably, the RSV pre-fusion F protein mutant comprises any one of mutations b1)-b40) compared to SEQ ID NO:3; Preferably, the RSV prefusion F protein mutant comprises an electrostatic mutation compared to SEQ ID NO: 3; Preferably, the RSV pre-fusion F protein mutant comprises any one of the mutations c1)-c18) compared to SEQ ID NO:

3.

3. The RSV prefusion F protein mutant according to any one of claims 1-2, characterized in that The RSV pre-fusion F protein mutant comprises a disulfide bond mutation and an electrostatic mutation compared to SEQ ID NO: 3; Preferably, the RSV prefusion F protein mutant comprises any one of the mutations d1)-d21) compared to SEQ ID NO: 3: d1) E487C, A490C and Q501R; d2) S180C, S186C and Q501R; d3) S180C, S186C, E487C, A490C and Q501R; d4) S55C, L188C, E487C, A490C and Q501R; d5) S155C, S290C and Q501R; d6) S 55C, L188C and Q501R; d7) S155C, S290C, E487C, A490C and Q501R; d8) E487C, A490C and N228K; d9) S180C, S186C and N228K; d10) S180C, S186C, E487C, A490C and N228K; d11) S55C, L188C, E487C, A490C and N228 K; d12) S155C, S290C and N228K; d13) S55C, L188C and N228K; d14) S155C, S290C, E487C, A490C and N228K; d15) E487C, A490C, N228K and Q501R; d16) S180C, S186C, N228K and Q501R; d17) S55C, L188C, N228K and Q501R; d18) S155C, S290C, E487C, A490C, N228K and Q501R; d19) S180C, S186C, E487C, A490C, N228K and Q501R; d20) S55C, L188C, E487C, A490C, N228K and Q501R; d21) S155C, S290C, N228K and Q501R; Preferably, the RSV prefusion F protein mutant comprises a disulfide bond mutation and a cavity filling mutation compared to SEQ ID NO: 3; Preferably, the RSV prefusion F protein mutant comprises any one of the mutations e1)-e28) compared to SEQ ID NO: 3: e1) E487C, A490C and V207L; e2) S180C, S186C and V207L; e3) S180C, S186C, E487C, A490C and V207L; e4) S55C, L188C, E487C, A490C and V207L; e5) S155C, S290C and V207L; e6) S55C, L188C and V207L; e7) S155C, S290C, E487C, A490C and V207L; e8) E487 C, A490C and V296I; e9) S180C, S186C and V296I; e10) S180C, S186C, E487C, A490C and V296I; e11) S55C, L188C, E487C, A490C and V296I; e12) S155C, S290C and V296I; e13) S55C, L188C and V296I; e14) S155C, S290C, E487C, A490C and V296I; e15) S55C, L188C, E487C, A490C, V207L ​​and L 334I; e16) S155C, S290C, V207L ​​and L334I; e17) S55C, L188C, V207L ​​and L334I; e18) S155C, S290C, E487C, A490C, V207L ​​and L334I; e19) ​​E487C, A490C, V296I and L334I; e20) S180C, S186C, V296I and L334I; e21) S180C, S186C, E487C, A490C, V296I and L334I; e22) S55C, L188C, E4 87C, A490C, V296I and L334I; e23) S155C, S290C, V296I and L334I; e24) S55C, L188C, V296I and L334I; e25) S155C, S290C, E487C, A490C, V296I and L334I; e26) E487C, A490C, V207L ​​and L334I; e27) S180C, S186C, V207L ​​and L334I; e28) S180C, S186C, E487C, A490C, V207L ​​and L334I; Preferably, the RSV prefusion F protein mutant comprises a disulfide bond mutation, a cavity filling mutation and an electrostatic mutation compared to SEQ ID NO: 3; Preferably, the RSV prefusion F protein mutant comprises any one of the mutations f1)-f84) compared to SEQ ID NO: 3: f1) E487C, A490C, V207L ​​and Q501R; f2) S180C, S186C, V207L ​​and Q501R; f3) S180C, S186C, E487C, A490C, V207L ​​and Q501R; f4) S55C, L188C, E487C, A490C, V207L ​​and Q501R; f5) S155C, S290C, V207L ​​and Q501R; f6) S180C, S186C, V296I and Q501R; f7) S180C, S186C, E487C, A490C, V296I and Q501R; f8) S55C, L188C, E487C, A490C, V296I and Q501R; f9) S155C, S290C, V296I and Q501R; f10) S55C, L188 C, V296I and Q501R; f11) S155C, S290C, E487C, A490C, V296I and Q501R; f12) E487C, A490C, V207L, L334I and Q501R; f13) S180C, S186C, V207L, L334I and Q501R; f1 4) S180C, S186C, E487C, A490C, V207L, L334I and Q501R; f15) S55C, L188C, E487C, A490C, V207L, L334I and Q501R; f16) S155C, S290C, V207L, L334I and Q501R; f 17) S55C, L188C, V207L, L334I and Q501R; f18) S155C, S290C, E487C, A490C, V207L, L334I and Q501R; f19) E487C, A490C, V296I, L334I and Q501R; f20) S180C, S1 86C, V296I, L334I and Q501R; f21) S180C, S186C, E487C, A490C, V296I, L334I and Q501R; f22) S155C, S290C, E487C, A490C, V296I, L334I and Q501R; f23) E487C, A490C, V207L ​​and N228K; f24) S180C, S186C, V207L ​​and N228K; f25) S180C, S186C, E487C, A490C, V207L ​​and N228K; f26) S55C, L188C, E487C, A490C, V207L ​​and N228K ; f27) S155C, S290C, V207L ​​and N228K; f28) S55C, L188C, V207L ​​and N228K; f29) S155C, S290C, E487C, A490C, V207L ​​and N228K; f30) E487C, A490C, V296I and N228K;f31) S180C, S186C, V296I and N228K; f32) S180C, S186C, E487C, A490C, V296I and N228K; f33) S55C, L188C, E487C, A490C, V296I and N228K; f34) S155C, S290C, V296I and N228K; f35) S55C, L188C, V296I and N228K; f36) S155C, S290C, E487C, A490C, V296I and N228K; f37) S155C, S290C, V207L, L334I and N228K; f38) S5 f39) S155C, S290C, E487C, A490C, V207L, L334I and N228K; f40) E487C, A490C, V296I, L334I and N228K; f41) S180C, S186C, V296I, L334I and N228K; f42) S180C, S186C, E487C, A490C, V296I, L334I and N228K; f43) S55C, L188C, E487C, A490C, V296I, L334I and N228K; f44) S155C, S 290C, V296I, L334I and N228K; f45) S55C, L188C, V296I, L334I and N228K; f46) S155C, S290C, E487C, A490C, V296I, L334I and N228K; f47) E487C, A490C, V207L, N228K and Q501R; f48) S180C, S186C, V207L, N228K and Q501R; f49) S180C, S186C, E487C, A490C, V207L, N228K and Q501R; f50) S55C, L188C, E487C, A490C, V207L, N228K and Q501R; f51) S155C, S290C, V207L, N228K and Q501R; f52) S55C, L188C, V207L, N228K and Q501R; f53) S180C, S186C, E487C, A490C, V296I, N228K and Q501R; f54) S55C, L188C, E487C, A490C, V296I, N228K and Q501R; f55) S155C, S290C, V296I, N228K and Q501R; f56) S55C, L188C, V296I, N228K and Q501R;f57) S155C, S290C, E487C, A490C, V296I, N228K and Q501R; f58) E487C, A490C, V207L, L334I, N228K and Q501R; f59) S180C, S186C, V207L, L334I, N228K and Q50 1R; f60) S180C, S186C, E487C, A490C, V207L, L334I, N228K and Q501R; f61) S55C, L188C, E487C, A490C, V207L, L334I, N228K and Q501R; f62) S155C, S290C, V2 07L, L334I, N228K and Q501R; f63) S55C, L188C, V207L, L334I, N228K and Q501R; f64) S155C, S290C, E487C, A490C, V207L, L334I, N228K and Q501R; f65) E487C, A 490C, V296I, L334I, N228K and Q501R; f66) S180C, S186C, V296I, L334I, N228K and Q501R; f67) S180C, S186C, E487C, A490C, V296I, L334I, N228K and Q501R; f68 )S55C, L188C, E487C, A490C, V296I, L334I, N228K and Q501R;; f69)E487C, A490C, V207L, L334I, and N228K; f70)S55C, L188C, V207L ​​and Q501R; f71)S155C, S29 0C, E487C, A490C, V207L ​​and Q501R; f72) E487C, A490C, V296I and Q501R; f73) S55C, L188C, E487C, A490C, V296I, L334I and Q501R; f74) S155C, S290C, V296I, L3 34I and Q501R; f75) S55C, L188C, V296I, L334I and Q501R; f76) S180C, S186C, V207L, L334I and N228K; f77) S180C, S186C, E487C, A490C, V207L, L334I and N228K; f 78) S55C, L188C, E487C, A490C, V207L, L334I and N228K; f79) S155C, S290C, E487C, A490C, V207L, N228K and Q501R; f80) E487C, A490C, V296I, N228K and Q501R;f81) S180C, S186C, V296I, N228K and Q501R; f82) S155C, S290C, V296I, L334I, N228K and Q501R; f83) S55C, L188C, V296I, L334I, N228K and Q501R; f84) S155C, S290C, E487C, A490C, V296I, L334I, N228K and Q501R. ; 4. A complex comprising the RSV pre-fusion F protein mutant according to any one of claims 1 to 3; and vector components capable of displaying said mutants; Preferably, the carrier component comprises at least one of nanomaterials, bacterial outer membrane vesicles (OMVs), polymerized bases, and virus-like particles (VLPs).

5. A fusion protein comprising the RSV pre-fusion F protein mutant according to any one of claims 1 to 3; and a trimerization base.

6. The fusion protein according to claim 5, characterized in that The amino acid sequence of the trimerization base is shown in SEQ ID NO:4; Preferably, the fusion protein comprises the RSV pre-fusion F protein mutant according to any one of claims 1 to 3 from the N-terminus to the C-terminus, and a trimerization base.

7. A recombinant protein comprising the fusion protein according to any one of claims 5 to 6; and optionally a tag sequence to facilitate expression and / or purification; Preferably, the tag sequence is selected from at least one of His tag, FLAG, Strep-Tag II, Poly arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, and NusA.

8. A biological material associated with the RSV pre-fusion F protein mutant according to any one of claims 1 to 3, the fusion protein according to any one of claims 5 to 6, or the recombinant protein according to claim 7, wherein the biological material comprises any one of n1) to n9): n1) a nucleic acid molecule encoding the RSV pre-fusion F protein mutant according to any one of claims 1 to 3, the fusion protein according to any one of claims 5 to 6, or the recombinant protein according to claim 7; n2) an expression cassette comprising the nucleic acid molecule described in n1); n3) a vector comprising the nucleic acid molecule described in n1); n4) a vector comprising the expression cassette described in n2); n5) a cell comprising the nucleic acid molecule described in n1); n6) a cell comprising the expression cassette described in n2); n7) a cell comprising the vector described in n3); n8) a cell comprising the vector described in n4); n9) a cell comprising the RSV pre-fusion F protein mutant according to any one of claims 1 to 3, the fusion protein according to any one of claims 5 to 6, or the recombinant protein according to claim 7; The cell of any one of n5) to n9) does not contain propagation material.

9. A conjugate comprising the RSV pre-fusion F protein mutant according to any one of claims 1 to 3, the fusion protein according to any one of claims 5 to 6, or the recombinant protein according to claim 7; and a coupling moiety, wherein the coupling moiety is a detectable label; Preferably, the detectable marker comprises at least one of metal particles, fluorescent markers, luminescent markers, electron-dense markers, chemiluminescent markers, radioactive markers, and enzyme markers.

10. Any application of (1)-(2); (1) Use of the RSV prefusion F protein mutant according to any one of claims 1 to 3, the complex according to claim 4, the fusion protein according to any one of claims 5 to 6, the recombinant protein according to claim 7, or the biomaterial according to claim 8 in any one of m1) to m2); m1) preparing RSV antibodies; m2) preparing medicines; The drug is used for 11) or 12): l1) Prevention of RSV infection; 12) Prevention and / or treatment of diseases caused by RSV infection; (2) Use of the RSV pre-fusion F protein mutant according to any one of claims 1 to 3, the fusion protein according to any one of claims 5 to 6, the recombinant protein according to claim 7, the biomaterial according to claim 8, or the conjugate according to claim 9 in the preparation of a kit; The kit is used for any one of o1)-o3): o1) detecting the presence or amount of RSV antibodies in a sample; o2) Diagnosis of RSV infection; o3) Diagnosis of diseases caused by RSV infection; Preferably, the disease caused by RSV infection includes at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

11. A kit comprising: the RSV pre-fusion F protein mutant according to any one of claims 1-3, the fusion protein according to any one of claims 5-6, the recombinant protein according to claim 7, or the conjugate according to claim 9.

12. A drug comprising: the RSV pre-fusion F protein mutant according to any one of claims 1 to 3, the complex according to claim 4, the fusion protein according to any one of claims 5 to 6, the recombinant protein according to claim 7, or the biomaterial according to claim 8.

13. The drug according to claim 12, characterized in that The drug further comprises: a pharmaceutically acceptable carrier; Preferably, the medicine further comprises: other active ingredients for preventing and / or treating RSV infection, or diseases caused by RSV infection.

14. A vaccine comprising: the RSV pre-fusion F protein mutant according to any one of claims 1 to 3, the complex according to claim 4, the fusion protein according to any one of claims 5 to 6, the recombinant protein according to claim 7, or the biological material according to claim 8; and adjuvants.

15. A method for preparing the RSV pre-fusion F protein mutant according to any one of claims 1 to 3, the fusion protein according to any one of claims 5 to 6, and the recombinant protein according to claim 7, which is obtained by culturing the cells according to claim 8.

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