RSV pre-fusion f protein mutant and use thereof

Abstract

The present invention relates to an RSV pre-fusion F protein mutant and the use thereof. The RSV pre-fusion F protein mutant has a relatively good stability and a relatively high expression level and / or immunogenicity, and can be used for preparing RSV antibodies, preventing RSV infections, preventing and / or treating diseases caused by RSV infections, detecting the presence or amount of RSV antibodies in a sample, diagnosing RSV infections, and diagnosing diseases caused by RSV infections.

Description

RSV fusion pre-fusion F protein mutant and its application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to RSV pre-fusion F protein mutants and their applications. Background Technology

[0002] Respiratory syncytial virus (RSV) was first discovered in 1956, and researchers have continuously attempted to develop live attenuated virus vaccines or inactivated whole virus vaccines. However, formalin-inactivated vaccines can exacerbate respiratory disease (ERD) during natural infection, and even today, the severe consequences of this event remain a crucial consideration in developing new vaccine methods and immunization strategies. With a deeper understanding of the pathogenesis of RSV and the vaccine-induced ERD effect, and with continuous trials and accumulated experience in developing monoclonal antibody drugs, live attenuated vaccines, nucleic acid vaccines, adenovirus vaccines, and subunit vaccines, the successful development of RSV vaccines is on the horizon.

[0003] The pre-fusion state of respiratory syncytial virus (RSV) fusion (F) glycoprotein is a major neutralizing target for antibodies in human serum, but its metastability hinders structural characterization of the pre-fusion state. The co-crystal structure resolution of an antibody D25 complexed with the F glycoprotein offers hope for vaccine design. Focusing on antigenic sites... The introduction of the DS-Cav vaccine design, which immobilizes the RSV fusion (F) glycoprotein in its pre-fusion state, offers better protection than previous vaccines. While existing technologies disclose the modification of the RSV-F protein using disulfide bond mutations, cavity-filling mutations, and electrostatic mutations, the stability and / or immunogenicity of the resulting mutants still need improvement. Therefore, it is necessary to develop a stable and / or immunogenic pre-fusion RSV F protein. Summary of the Invention

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

[0005] A second aspect of the present invention is to provide a composite.

[0006] A third aspect of the present invention is to provide a fusion protein.

[0007] The fourth aspect of this invention is to provide a recombinant protein.

[0008] The fifth aspect of this invention aims to provide biomaterials related to the mutants of the first aspect, the fusion proteins of the third aspect, and the recombinant proteins of the fourth aspect.

[0009] The sixth aspect of this invention is to provide a coupling material.

[0010] The seventh aspect of this invention aims to provide applications of the mutants of the first aspect, the complexes of the second aspect, the fusion proteins of the third aspect, the recombinant proteins of the fourth aspect, the biomaterials of the fifth aspect, and the conjugates of the sixth aspect.

[0011] An eighth aspect of the present invention is to provide a reagent kit.

[0012] The object of the ninth aspect of this invention is to provide a medicine.

[0013] The tenth aspect of this invention is to provide a vaccine.

[0014] The eleventh aspect of this invention aims to provide methods for preparing the mutant of the first aspect, the fusion protein of the third aspect, and the recombinant protein of the fourth aspect.

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

[0016] The object of the thirteenth aspect of this invention is to provide a method.

[0017] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In the first aspect of the present invention, a pre-fusion F protein mutant of respiratory syncytial virus (RSV) is provided, wherein the pre-fusion F protein mutant of RSV, compared with SEQ ID NO: 3, contains at least one mutation of (1)-(3) (the resulting mutant is called pre-fusion F protein mutant of respiratory syncytial virus (RSV) subtype A): or the pre-fusion F protein mutant of RSV, compared with SEQ ID NO: 29, contains at least one mutation of (1)-(3) (the resulting mutant is called pre-fusion F protein mutant of respiratory syncytial virus (RSV) subtype B); (1) disulfide bond mutation; (2) cavity filling mutation; (3) electrostatic mutation; The disulfide bond mutation includes any one of the mutations 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 S50 2C; a12) E487C, A490C, G242C and M289C; a13) E487C, A490C, T54C 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, T10 3C and I148C; a19) E487C, A490C, R106C and V144C; a20) E487C, A490C, L138C and T337C; a21) E487C, A490C, G139C and Q354C; a22) E487C, A490C, L142C and N371C; a23) E487C, A490C, G145C and Q370C; a24) E487C, A490C, G151C and Q302C; a25) E487C a26) E487C, A490C, L160C and E163C; a27) E487C and A490C, G162C and N165C; a28) E487C, A490C, A170C and A177C; a29) E487C, A490C, S180C and S186C; a30) E487C, A490C, N105C and A147C; a31) E487C, A490C, G151C and V302C; a32) 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 includes any one of the mutations 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) 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; The electrostatic mutation includes any one of the mutations 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.

[0018] In some embodiments, the RSV pre-fusion F protein mutant contains a disulfide bond mutation compared to SEQ ID NO: 3 or SEQ ID NO: 29.

[0019] In some embodiments, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3 or SEQ ID NO: 29, contains any one of the mutations a1)-a38); further includes the mutation described in a28).

[0020] In some embodiments, the RSV pre-fusion F protein mutant contains a cavity-filling mutation compared to SEQ ID NO: 3 or SEQ ID NO: 29.

[0021] In some embodiments, the RSV pre-fusion F protein mutant contains any one of the mutations b1)-b40) compared to SEQ ID NO: 3 or SEQ ID NO: 29.

[0022] In some embodiments, the RSV pre-fusion F protein mutant contains electrostatic mutations compared to SEQ ID NO: 3 or SEQ ID NO: 29.

[0023] In some embodiments, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3 or SEQ ID NO: 29, contains any one of the mutations c1)-c18); further contains any one of the mutations c17)-c18).

[0024] In some embodiments, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3 or SEQ ID NO: 29, contains disulfide bond mutations and electrostatic mutations.

[0025] In some embodiments, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3 or SEQ ID NO: 29, includes any one of the mutations d1)-d21): 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 With Q501R; d18) S155C, S290C, E487C, A490C, N228K with Q501R; d19) S180C, S186C, E487C, A490C, N228K with Q501R; d20) S55C, L188C, E487C, A490C, N228K with Q501R; d21) S155C, S290C, N228K with Q501R.

[0026] In some embodiments, the RSV fusion-pre-fusion F protein mutant, compared to SEQ ID NO: 3 or SEQ ID NO: 29, contains disulfide bond mutations and cavity-filling mutations.

[0027] In some embodiments, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3 or SEQ ID NO: 29, includes any one of the mutations e1)-e28): 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, S186C, 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 With 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 including any one of the mutations in e4) and e11).

[0028] In some embodiments, the RSV fusion-pre-fusion F protein mutant, compared to SEQ ID NO: 3 or SEQ ID NO: 29, includes disulfide bond mutations, cavity-filling mutations, and electrostatic mutations.

[0029] In some embodiments, the RSV pre-fusion F protein mutant is associated with SEQ ID NO: 3 or SEQ ID NO: 3. Compared to NO:29, it includes any one of the mutations in 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, S186CV296I 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 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, V296I and N228K; f33) S55C, L188C, E487C, A490C, V296I and N228K; f34) S155C, S290C 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, L334I 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, 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, N2 28K 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 Q501R; f60) S1 80C, 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; f65) 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 f72) E487C, A490C, V207L ​​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;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 Q501R; f83) S55C, L188C, V296I, L334I, N228K and Q501R; f84) S155C, S290C, E487C, A490C, V296I, L334I, N228K and Q501R; further including any one of the mutations f7), f14), f23), f25), f26), f47), f49), f54), f60), f78); and further including any one of the mutations f14), f49), f60).

[0030] A second aspect of the invention provides a complex comprising the RSV pre-fusion F protein mutant of the first aspect of the invention; and a vector component capable of displaying said mutant.

[0031] In some embodiments, the carrier component comprises at least one of nanomaterials, bacterial outer membrane vesicles (OMVs), polymerized pedestals, and virus-like particles (VLPs).

[0032] In some embodiments, the nanomaterial comprises at least one of lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers, and biomimetic nanoparticles.

[0033] In some embodiments, the VLP is assembled from proteins derived from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

[0034] A third aspect of the invention provides a fusion protein comprising the RSV pre-fusion F protein mutant of the first aspect of the invention; and a trimerization base.

[0035] In some embodiments, the amino acid sequence of the trimerization site is shown in SEQ ID NO: 4.

[0036] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, an RSV pre-fusion F protein mutant of the first aspect of the invention, and a trimerization base.

[0037] A fourth aspect of the invention provides a recombinant protein comprising the fusion protein of the third aspect of the invention; and optionally a tag sequence to assist in expression and / or purification.

[0038] In some embodiments, 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.

[0039] In some embodiments, the recombinant protein comprises, from the N-terminus to the C-terminus, a fusion protein according to the third aspect of the invention, and optionally a tag sequence to assist in expression and / or purification.

[0040] A fifth aspect of the present invention provides biological materials relating to 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, said biological materials comprising any one of n1)-n9): 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; n2) an expression cassette comprising the nucleic acid molecule of n1); n3) a vector comprising the nucleic acid molecule of n1); n4) a vector comprising the expression cassette of n2); n5) a cell comprising the nucleic acid molecule of n1); n6) a cell comprising the expression cassette of n2); n7) a cell comprising the vector of n3); n8) a cell comprising the vector of n4); 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.

[0041] In some embodiments, any of the cells described in n5)-n9) does not contain reproductive material.

[0042] In some embodiments, any of the vectors n3)-n4) includes a prokaryotic expression vector and a eukaryotic expression vector.

[0043] In some embodiments, the eukaryotic expression vector includes yeast expression vectors, mammalian expression vectors, insect expression vectors, etc.

[0044] In some embodiments, any one of the cells (n5)-n9) is selected from prokaryotic cells and eukaryotic cells.

[0045] In some embodiments, the prokaryotic cells include bacterial cells, Escherichia coli, and Streptomyces.

[0046] In some embodiments, the eukaryotic cells include yeast cells, mammalian cells, insect cells, etc.

[0047] In some embodiments, the mammal is selected from humans, monkeys, mice, rats, hamsters, goats, sheep, cattle, pigs, dogs, and cats.

[0048] A sixth aspect of the present invention provides a conjugate comprising an RSV pre-fusion F protein mutant of the first aspect of the present invention, a fusion protein of the third aspect, or a recombinant protein of the fourth aspect; and a conjugate portion, said conjugate portion being a detectable marker.

[0049] In some embodiments, the detectable marker includes at least one of metal particles, fluorescent markers, luminescent markers, electron-dense markers, chemiluminescent markers, radioactive markers, and enzyme markers.

[0050] In some embodiments, the detectable marker comprises at least one of a radioisotope, a fluorophore, luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose-6-phosphate dehydrogenase, rhodamine, luciferin, glucose oxidase, galactose oxidase, acridinium ester, acridinium sulfonamide, luminol, and isoluminol.

[0051] 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.

[0052] A seventh aspect of the invention provides the use of the RSV pre-fusion F protein 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, or the biological material of the fifth aspect in any one of m1)-m2); m1) preparing an RSV antibody; m2) preparing a drug; said drug for 11) or 12): l1) preventing RSV infection; l2) preventing and / or treating diseases caused by RSV infection.

[0053] The application of 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, the biomaterial of the fifth aspect, or the conjugate of the sixth aspect in the preparation of a kit; the kit is used for any one of o1)-o3): o1) detecting the presence or content of RSV antibody in a sample; o2) diagnosing RSV infection; o3) diagnosing diseases caused by RSV infection.

[0054] In some implementations, the RSV includes at least one of RSV A subtype and RSV B subtype.

[0055] In some embodiments, the RSV comprises an RSV A subtype (when the RSV pre-fusion F protein mutant comprises an RSV A subtype pre-fusion F protein mutant).

[0056] In some embodiments, the RSV comprises the RSV B subtype (when the RSV pre-fusion F protein mutant comprises the RSV B subtype pre-fusion F protein mutant).

[0057] In some embodiments, the diseases caused by the RSV infection include at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

[0058] An eighth aspect of the present invention provides a kit comprising: an RSV pre-fusion F protein mutant of the first aspect of the present invention, a fusion protein of the third aspect, a recombinant protein of the fourth aspect, or a conjugate of the sixth aspect.

[0059] In some embodiments, the kit is used for any one of o1)-o3): o1) detecting the presence or content of RSV antibodies in a sample; o2) diagnosing RSV infection; o3) diagnosing diseases caused by RSV infection.

[0060] In some embodiments, the RSV described in o1)-o3) includes at least one of RSV subtype A and RSV subtype B.

[0061] In some embodiments, the RSV described in o1)-o3) includes the RSV A subtype (when the RSV pre-fusion F protein mutant includes the RSV A subtype pre-fusion F protein mutant).

[0062] In some embodiments, the RSV described in o1)-o3) includes the RSV B subtype (when the RSV pre-fusion F protein mutant includes the RSV B subtype pre-fusion F protein mutant).

[0063] In some embodiments, the diseases caused by the RSV infection include at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

[0064] A ninth aspect of the present invention provides a medicament comprising: an RSV pre-fusion F protein mutant of the first aspect of the present invention, a complex of the second aspect, a fusion protein of the third aspect, a recombinant protein of the fourth aspect, or a biological material of the fifth aspect.

[0065] In some embodiments, the drug further comprises a pharmaceutically acceptable carrier.

[0066] In some embodiments, the medicament further comprises: other active ingredients for the prevention and / or treatment of RSV infection, or diseases caused by RSV infection.

[0067] In some embodiments, the drug is used for l1) or l2): l1) prevention and / or treatment of RSV infection; l2) prevention and / or treatment of diseases caused by RSV infection.

[0068] In some implementations, the RSV includes at least one of RSV A subtype and RSV B subtype.

[0069] In some embodiments, the RSV comprises an RSV A subtype (when the RSV pre-fusion F protein mutant comprises an RSV A subtype pre-fusion F protein mutant).

[0070] In some embodiments, the RSV comprises the RSV B subtype (when the RSV pre-fusion F protein mutant comprises the RSV B subtype pre-fusion F protein mutant).

[0071] In some embodiments, the diseases caused by the RSV infection include at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

[0072] A tenth aspect of the present invention provides a vaccine comprising: an RSV pre-fusion F protein mutant of the first aspect of the present invention, a complex of the second aspect, a fusion protein of the third aspect, a recombinant protein of the fourth aspect, or a biological material of the fifth aspect; and an adjuvant.

[0073] In some embodiments, the vaccine is used for l1) or l2): l1) prevention and / or treatment of RSV infection; l2) prevention and / or treatment of disease caused by RSV infection.

[0074] In some embodiments, the RSV described in l1)-l2) includes at least one of RSV subtype A and RSV subtype B.

[0075] In some embodiments, the RSV described in l1)-l2) includes the RSV A subtype (when the RSV pre-fusion F protein mutant includes the RSV A subtype pre-fusion F protein mutant).

[0076] In some embodiments, the RSV described in l1)-l2) includes the RSV B subtype (when the RSV pre-fusion F protein mutant includes the RSV B subtype pre-fusion F protein mutant).

[0077] In some embodiments, the diseases caused by the RSV infection include at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

[0078] 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, obtained by culturing the cells of the fifth aspect of the present invention.

[0079] The twelfth aspect of the present invention provides a method for preventing RSV infection or diseases caused by RSV infection, by administering an effective amount of the drug of the ninth aspect of the present invention or the vaccine of the tenth aspect of the present invention to a subject.

[0080] In some implementations, the RSV includes at least one of RSV A subtype and RSV B subtype.

[0081] In some embodiments, the RSV comprises an RSV A subtype (when the RSV pre-fusion F protein mutant comprises an RSV A subtype pre-fusion F protein mutant).

[0082] In some embodiments, the RSV comprises the RSV B subtype (when the RSV pre-fusion F protein mutant comprises the RSV B subtype pre-fusion F protein mutant).

[0083] In some embodiments, the diseases caused by the RSV infection include at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

[0084] In some implementations, the subject may include mammals, such as humans or non-human mammals.

[0085] In some implementations, the non-human mammals may include, but are not limited to, non-human primates (e.g., monkeys, orangutans), mice, rats, hamsters, gerbils, cats, dogs, guinea pigs, rabbits, horses, sheep, cattle, pigs, etc.

[0086] 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; said method is used for any one of o1)-o3): o1) detecting the presence or content of RSV antibodies in a sample; o2) diagnosing RSV infection; o3) diagnosing a disease caused by RSV infection.

[0087] In some embodiments, the RSV described in o1)-o3) includes at least one of RSV subtype A and RSV subtype B.

[0088] In some embodiments, the RSV described in o1)-o3) includes the RSV A subtype (when the RSV pre-fusion F protein mutant includes the RSV A subtype pre-fusion F protein mutant).

[0089] In some embodiments, the RSV described in o1)-o3) includes the RSV B subtype (when the RSV pre-fusion F protein mutant includes the RSV B subtype pre-fusion F protein mutant).

[0090] In some embodiments, the diseases caused by the RSV infection include at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

[0091] The beneficial effects of this invention are: This invention provides a pre-fusion F protein mutant of respiratory syncytial virus (RSV), 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 samples, diagnose RSV infection, and diagnose diseases caused by RSV infection. Attached Figure Description

[0092] Figure 1 shows the neutralizing titer NT50 of RSV in the serum of mice immunized with the mutant.

[0093] Figure 2 shows the neutralizing titer NT50 of RSV in the serum of mice immunized with the RSV B subtype fusion pre-F protein mutant. Detailed Implementation

[0094] The present invention will be further described in detail below through specific embodiments.

[0095] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0096] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0097] Example 1. Construction of a plasmid expressing the RSV pre-fusion F protein mutant (RSV A subtype pre-fusion F protein mutant): Based on the plasmid expressing wild-type RSV-F protein (containing the extracellular domain, locus fold, and His tag of wild-type RSV-F protein (wild-type RSV A subtype pre-fusion F protein); WT) (WT-F plasmid), point mutations were continuously introduced by PCR, and homologous recombination was used to complete the construction of the plasmid expressing the RSV pre-fusion F protein mutant (containing the extracellular domain mutant, locus fold, and His tag of RSV pre-fusion F protein). The plasmid expressing wild-type RSV-F protein was constructed by Sangon Biotech (Shanghai) Co., Ltd. (the codon-optimized nucleotide sequence (optimized according to human standards) was inserted into pCDNA3.1(+) through two restriction enzyme sites, NheI and BamHI). The codon-optimized nucleotide sequence is as follows: (5'-3', SEQ ID NO: 1), its nucleotides 1-6 are the kozak sequence, nucleotides 7-1545 are the coding sequence for the extracellular domain of the wild-type RSV-F protein, nucleotides 1546-1656 are the coding sequence for the base fold, and nucleotides 1657-1680 are the coding sequence for the His tag; its encoded amino acid sequence is: (N-terminal-C-terminal, SEQ ID NO: 2), amino acids 1-513 are the extracellular domain of the wild-type RSV-F protein (numbered SEQ ID NO: 3), amino acids 514-550 are the base fold (numbered SEQ ID NO: 4), and amino acids 551-558 are the His tag).

[0098] 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.

[0099] The plasmids of RSV pre-fusion 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 were constructed, and the PCR reaction system and homologous recombination process were used as an example: (1) DS-CAV construction: ① In the first round, WT-F plasmid was used as a template. 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 recombinated using the reaction system in Table 3. ② The second round of PCR used the plasmid constructed in ① as a template. 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 used in the reaction system in Table 3 for homologous recombination. (2) Construction of hd1b-1: ① The first round used WT-F plasmid as a template. 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 used in the reaction system in Table 3 for homologous recombination. ② The second round of PCR used the plasmid constructed in ① as a 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, and other reagents are shown in Table 1. The PCR products were used in the reaction system in Table 3 for homologous recombination. (3) Construction of hd1c-3: Using the hd1b-1 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, and other reagents are shown in Table 1. The PCR products were used in the reaction system in Table 3 for homologous recombination. (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-R; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were used for 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-R; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were used in the reaction system in Table 3 for homologous recombination. (6) Construction of hd4-2: ① In the first round, WT-F plasmid was used as template. 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 used in the reaction system in Table 3 for homologous recombination. ② The second round of PCR used the plasmid constructed in ① as a template. 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 used in the reaction system in Table 3 for homologous recombination. (7) Construction of hd4b-1: Using the hd4-2 plasmid as a 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, and other reagents are shown in Table 1. The PCR products were used in the reaction system in Table 3 for homologous recombination. (8) Construction of he1c-1: ① In the first round of PCR, WT-F plasmid was used as a template. Short PCR fragments were amplified using primers (V296I-F; E487C, A490C-R), and long fragments were amplified using primers (V296I-R; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were used in the reaction system shown in Table 3 for homologous recombination. ② In the second round of PCR, the plasmid constructed in ① was used as a template. Short PCR fragments were amplified using primers (N228K-F; Q501R-R), and long fragments were 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 used in the reaction system shown in Table 3 for homologous recombination. (9) Construction of he3a-3: Using plasmid (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 recombinated using the reaction system in Table 3.(10) Construction of he3b-3: Using plasmid (8)① as a 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 used in the reaction system in Table 3 for homologous recombination. (11) Construction of he4-1: Using plasmid (8)① as a template, 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 used in the reaction system in Table 3 for homologous recombination. (12) Construction of he4c-1: Using he4-1 plasmid as template, the primers for the short PCR fragment were (N228K-F; Q501R-R), and the primers for the long PCR fragment were (N228K-R; Q501R-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were used in the reaction system in Table 3 for homologous recombination. (13) Construction of hf3-3: ① In the first round, WT-F plasmid was used as template. 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 used in the reaction system in Table 3 for homologous recombination. ② The second round of PCR used the plasmid constructed in ① as a 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-R; E487C, A490C-F). The primer sequences are shown in Table 2, and other reagents are shown in Table 1. The PCR products were used in the reaction system in Table 3 for homologous recombination. (14) hf3c-1 construction: 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, and other reagents are shown in Table 1. The PCR products were used in the reaction system in Table 3 for homologous recombination. (15) Construction of hf3a-1: Using hf3-3 plasmid as 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, and other reagents are shown in Table 1. The PCR products were homologous recombination using the reaction system in Table 3.(16) Construction of hf4b-1: ① In the first round of PCR, plasmid (13)① was used as a 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, and other reagents are shown in Table 1. The PCR products were used in the reaction system in Table 3 for homologous recombination. ② In the second round of PCR, plasmid constructed in ① was used as a template. 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 used in the reaction system in Table 3 for homologous recombination. (17) Construction of hg1b-1: ① In the first round of PCR, WT-F plasmid was used as a template. Short PCR fragments were amplified using primers (V207L-F; E487C, A490C-R), and long fragments were 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 used in the reaction system shown in Table 3 for homologous recombination. ② In the second round of PCR, the plasmid constructed in ① was used as a template. Short PCR fragments were amplified using primers (V207L-F; V296I-R), and long fragments were 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 used in the reaction system shown in Table 3 for homologous recombination.

[0100] Table 1 PCR reaction system

[0101] Table 2 Primer sequences

[0102] Table 3. Homologous recombination reaction system and conditions

[0103] The constructed plasmid expressing the RSV pre-fusion F protein mutant was transformed into E. coli DH5α competent cells, plated in solid LB broth (final Amp concentration 100 μg / mL), cultured overnight, and then amplified in liquid LB (final Amp concentration 100 μg / mL). The cells were then sequenced and identified. The plasmid expressing the RSV pre-fusion F protein mutant (the amino acid sequence of the protein expressed by the plasmid expressing the RSV pre-fusion F protein mutant compared with the mutation sites in SEQ ID NO: 2 is shown in Table 4) was successfully constructed.

[0104] Table 4. Mutation sites corresponding to each mutant.

[0105] 2. Stability testing of mutants: The stability of mutants was tested as follows: 1) A 96-well plate was used, with 1x10 μL of material per well. 4 1) 100 μL of 293t cells were seeded into plates. On the second day, the plasmid of the pre-fusion F protein mutant of RSV was transfected with PEI (100 ng / well). On the third day, 100 μL of medium containing 10% FBS was added, and the cells were cultured for another 4 days. 2) The cell culture supernatants with the same density obtained in 1) were treated as follows: placed at 4°C for 1 week (4°C-1w), at 50°C for 1 hour (50°C-1h), and / or at 60°C for 1 hour (60°C-1h), and then subjected to sandwich ELISA detection.

[0106] The sandwich ELISA detection method is as follows: 1) Coat the cells overnight with 100 ng / well of D25 (Biodragon, BD-VA1156) or AM14 (Biodragon, BD-VA1155) antibody; 2) Dilute the treated or untreated cell culture supernatant 5-125 times or not (see Table 5-10 for specific dilutions), add 100 μL / well of the treated or untreated cell culture supernatant to a 96-well plate coated with antibody after washing 3 times with PBST, and incubate overnight; 3) After washing 3 times with PBST, add 50 ng / well of anti-His antibody (Proteintech, 66005-1-G-1000UL), and incubate at room temperature for 1.5 h; 4) After washing 3 times with PBST, add 100 ng / well of mouse secondary antibody with HRP (Jackson Immunosorbent Assay). research, 115-035-146), room temperature 1h; 5) wash 3 times with PBST, treat with ELISA developing solution for 15min, and read the OD450 value immediately after termination with stop solution.

[0107] The stability results of the mutants are shown in Tables 5, 6, 7, 8, 9, and 10 (due to the stability analysis being conducted in different batches, some mutant results are duplicated): The above mutants exhibit 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, and 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, he1c-1 are superior to or equivalent to DS-CAV (at least under 50℃-1h treatment).

[0108] Table 5. Results of single mutations in disulfide bonds (5-fold dilution)

[0109] Note: "D25" indicates that the D25 antibody test was used.

[0110] Table 6. Results of disulfide bond double mutagenesis (5-fold dilution)

[0111] Note: "D25" indicates that the D25 antibody test was used.

[0112] Table 7. Results of electrostatic single mutations (5-fold dilution)

[0113] Note: "D25" indicates that the D25 antibody test was used.

[0114] Table 8. Results of single mutations in the cavity (undiluted)

[0115] Note: "D25" indicates that the D25 antibody test was used.

[0116] Table 9. Results of electrostatic / cavitary double mutation (5-fold dilution)

[0117] Note: "D25" indicates that the D25 antibody test was used.

[0118] Table 10 Results of three types of combined mutations (n=3) (125-fold dilution)

[0119] Note: "D25" indicates that D25 antibody detection was used, and "AM14" indicates that AM14 antibody detection was used.

[0120] 3. RSV-F Affinity Assay with Related Neutralizing Antibodies: From 133 combinations of disulfide bond mutations, cavity-filling mutations, and electrostatic mutations (Table 10), 36 mutant combinations (Table 11) were selected for affinity testing: 1) When the cell density reached 2x10 at 293f... 6 / 100μL, transfected with PEI (transfection plasmid amount 2μg / mL); 2) Collect the cell suspension, centrifuge at 1000rpm for 10min to remove cells, centrifuge at 8000rpm for 30min to remove cell debris, and filter through a 0.45μm pore size filter; 3) Use Ultra filter, 30kDa MWCO (UFC5030), concentrated filtrate, and replaced buffer with PBS; 4) Use Biacore 8K surface plasmon resonance (SPR) to test the affinity of the above mutants for neutralizing antibodies D25 (Biodragon, BD-VA1156), AM14 (Biodragon, BD-VA1155), AM22 (Biodragon, BD-VA1358), 101F (AntibodySystem, AntibodySystem_RVV02816), and MOTE (Prosci, 10-089) (refer to the instruction manual for detection methods).

[0121] The results are shown in Table 11: the above mutants have good affinity for the neutralizing antibody against RSV-F.

[0122] Table 11 Results of antigen epitope affinity

[0123] 4. Expression Level Testing: Based on the combined data from thermostability and affinity tests, 16 mutant proteins (Table 12) were screened for expression level testing. 1) The expression levels of some mutants were detected as follows: 1 x 10⁻⁶ protein per well in a 96-well plate. 4 293t cells were seeded at 100 μL each and transfected with plasmid using PEI (100 ng / well) the next day. On the third day, 100 μL of medium containing 10% FBS was added, and the cells were cultured for another 4 days. The secreted F protein in the cell culture supernatant was detected using a sandwich ELISA method (the method is the same as in "2. Detection of mutant stability"). The results are shown in Table 12: The expression levels of mutants hd1b-1, hd1c-3, hd3b-1, hd3c-2, hd4-2, hd4b-1, helc-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 comparable to those of DS-CAV.

[0124] Table 12 Expression levels of some mutants (n=3)

[0125] Note: "D25" indicates that the D25 antibody test was used.

[0126] 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: 1) At a cell density of 2x10⁻⁶ cells / 293f. 6 1) Transfect 100 μL of the protein sample with PEI (transfection plasmid amount: 2 μg / mL); 2) 6 days after transfection, collect the cell suspension and centrifuge at 1000 rpm for 10 min and 8000 rpm for 30 min to remove cells and cell debris, obtaining cell supernatant; 3) Filter the supernatant again using vacuum filtration to prevent clogging of the purification column; 4) Add a nickel column to the purification column and perform column pass-through of the supernatant three times, followed by elution; 5) Concentrate the eluted protein solution using a concentration tube and replace the buffer with 1XPBS; 6) Determine the protein concentration using the BCA protein assay method; 7) Take 10 μL of the protein sample after the protein concentration test, add 5x loading buffer, and heat denature at 98℃ for 10 min, followed by SDS-PAGE gel electrophoresis; 8) Use Coomassie blue to stain the protein to determine protein purity.

[0127] 6. Immunization of mice: Five Balb / c mice were immunized with each protein antigen. 50 μL of the protein solution obtained in step 5 (protein concentration of 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 day 21. Blood was collected from the eyeballs on day 35, and serum was separated for subsequent neutralization experiments.

[0128] 7. Neutralization Experiment 1) Use culture medium containing 2% fetal bovine serum at a ratio of 1 x 10⁻⁶ cells / well. 4 1) Coat 100 μL of Hep2 cells into plates; 2) Dilute the mouse serum obtained in step 6 to 10-fold, and then perform serial dilutions of 3-fold (using 100 TCID50 RSV virus solution) (GenBank: MW582527.1), for a total of 7 dilutions. After mixing, incubate at 37°C for 1 hour, inverting and shaking once during the process; 3) Add 100 μL of the diluted serum from step 2) to each well of the plate in step 1), with 6 replicates per dilution. Set up positive control (400 TCID50 RSV virus solution) and negative control (medium containing 2% fetal bovine serum); 4) After two days of culture, remove the supernatant and wash twice with PBS, add 4% paraformaldehyde, and seal with sealant; 5) Use high-content scanning plate to count the fluorescence area of ​​each well and perform neutralization data analysis; 6) Use the built-in analysis function of GraphPad Prism to calculate the neutralization NT50.

[0129] The results are shown in Figure 1: 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.

[0130] 8. Similar effects can be achieved by mutations at the same site in the F protein of RSV B isotype. Based on a plasmid expressing the F protein of wild-type RSV B isotype (WT-F(B) plasmid), point mutations were continuously introduced via PCR, and homologous recombination was used to construct a plasmid expressing the pre-fusion F protein mutant of RSV B isotype. The WT-F(B) plasmid differs from the WT-F plasmid in that the coding sequence of the extracellular domain of the wild-type RSV-F protein in the WT-F plasmid was replaced with the coding sequence of the extracellular domain of the wild-type B isotype RSV-F protein. The amino acid sequence of the extracellular domain of the wild-type B isotype RSV-F protein is as follows: N-terminus-C-terminus, SEQ ID NO: 29; The constructed plasmid expressing the RSV B subtype pre-fusion F protein mutant was transformed into E. coli DH5α competent cells, plated in solid LB broth (final Amp concentration 100 μg / mL), cultured overnight, and then amplified in liquid LB broth (final Amp concentration 100 μg / mL). Sequencing was performed to identify the plasmid expressing the RSV B subtype pre-fusion F protein mutant (the amino acid sequence of the protein expressed by the plasmid expressing the RSV B subtype pre-fusion F protein mutant compared to the wild-type RSV B subtype F protein...). The N-terminal-C-terminal structure (SEQ ID NO: 30, amino acids 1-513 are the extracellular domain of wild-type subtype B RSV-F protein (SEQ ID NO: 29), amino acids 514-550 are the base fold, and amino acids 551-558 are the His tag) contains mutation sites as shown in Table 13. The structure was successfully constructed.

[0131] Table 13 Loci corresponding to each mutant

[0132] Note: ABRYSVO-B is RSV preF B in Pfizer's respiratory syncytial virus (RSV) vaccine Abrysvo (RSVpreF).

[0133] The immunogenicity of the RSV B subtype pre-fusion F protein mutant was verified according to the following steps: “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”; “6. Mouse immunization”; and “7. Neutralization experiment”. (In the neutralization experiment, the virus (GenBank: MW582527.1) was replaced with another virus (GenBank: MW582529). The results are shown in Figure 2. Similar to the RSV pre-fusion F protein mutant in “7. Neutralization experiment”, the neutralization effect was superior to that of wild-type RSV.) The B subtype of RSV-F protein (WT-B) is even superior to DS-CAV-B, especially hd3c-B and hf3c-B, which are superior to Pfizer's ABRYSVO-B. It can be seen that the mutations in the wild-type B subtype RSV-F protein shown in Table 4 above have similar effects to those in the wild-type A subtype RSV-F protein shown in Table 4 above, with better stability, better affinity for related antibodies, higher expression levels and / or immunogenicity.

[0134] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A pre-fusion mutant of respiratory syncytial virus (RSV) F protein, wherein the pre-fusion mutant of RSV F protein, compared with SEQ ID NO: 3, contains at least one mutation of (1)-(3): or The RSV fusion-pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains at least one mutation among (1)-(3); (1) disulfide bond mutation; (2) cavity filling mutation; (3) electrostatic mutation; The disulfide bond mutations include any one of the mutations 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 1199C; 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 includes any one of the mutations 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 includes any one of the mutations 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 pre-fusion F protein mutant according to claim 1, characterized in that, The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3, contains a disulfide bond mutation; or The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains a disulfide bond mutation; Preferably, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3, contains any one of the mutations a1)-a38); or The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains any one of the mutations a1)-a38); Preferably, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3, contains a cavity-filling mutation; or The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains a cavity-filling mutation; Preferably, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3, contains any one of the mutations b1)-b40); or The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains any one of the mutations b1)-b40); Preferably, the RSV pre-fusion F protein mutant contains an electrostatic mutation compared to SEQ ID NO: 3; or The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains electrostatic mutations; Preferably, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3, contains any one of the mutations c1)-c18); or The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains any one of the mutations c1)-c18).

3. The RSV pre-fusion F protein mutant according to any one of claims 1-2, characterized in that, The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3, contains disulfide bond mutations and electrostatic mutations; or The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains disulfide bond mutations and electrostatic mutations; Preferably, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3, includes any one of the mutations d1)-d21): 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; or The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains any one of the mutations d1)-d21): 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 With Q501R; d18) S155C, S290C, E487C, A490C, N228K with Q501R; d19) S180C, S186C, E487C, A490C, N228K with Q501R; d20) S55C, L188C, E487C, A490C, N228K with Q501R; d21) S155C, S290C, N228K with Q501R; Preferably, the RSV pre-fusion F protein mutant, compared to SEQ ID NO: 3, contains a disulfide bond mutation and a cavity-filling mutation; or The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains disulfide bond mutations and cavity-filling mutations; preferably, the RSV pre-fusion F protein mutant compared to SEQ ID NO: 29... Compared to NO:3, it includes any one of the mutations from e1) to e28): 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 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 L3 34I; 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, E48 7C, 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; or The RSV pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains any one of the mutations e1)-e28): 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 pre-fusion F protein mutant, compared to SEQ ID NO: 3, contains disulfide bond mutations, cavity-filling mutations, and electrostatic mutations; or The RSV fusion-pre-fusion F protein mutant, compared to SEQ ID NO: 29, contains disulfide bond mutations, cavity-filling mutations, and electrostatic mutations; Preferably, the RSV pre-fusion F protein mutant, compared with SEQ ID NO: 3, contains 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 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; 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 5C, 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, L334I and N228K; f43) S55C, L188C, E487C, A490C, V296I, L334I and N228K; f44) S155C, S 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 f69) S55C, L188C, E487C, A490C, V296I, L334I, N228K and Q501R; f70) E487C, A490C, V207L, L334I and N228K; f71) S55C, L188C, V207L ​​and Q501R; f72) 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; or; Compared with SEQ ID NO: 29, the RSV pre-fusion F protein mutant contains 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 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; 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 5C, 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, L334I and N228K; f43) S55C, L188C, E487C, A490C, V296I, L334I and N228K; f44) S155C, S 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 f69) S55C, L188C, E487C, A490C, V296I, L334I, N228K and Q501R; f70) E487C, A490C, V207L, L334I and N228K; f71) S55C, L188C, V207L ​​and Q501R; f72) 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 1-3; And the vector components that can display the mutant; Preferably, the carrier component comprises at least one of nanomaterials, bacterial outer membrane vesicles (OMVs), polymerized pedicles, and virus-like particles (VLPs).

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

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

7. A recombinant protein comprising the fusion protein according to any one of claims 5-6; And optional tag sequences to assist in 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 biomaterial relating to the RSV pre-fusion F protein mutant of any one of claims 1-3, the fusion protein of any one of claims 5-6, or the recombinant protein of claim 7, wherein the biomaterial comprises any one of n1)-n9): n1) A nucleic acid molecule encoding the RSV pre-fusion F protein mutant as described in any one of claims 1-3, the fusion protein as described in any one of claims 5-6, or the recombinant protein as described in claim 7; n2) contains an expression cassette containing the nucleic acid molecule described in n1); n3) A carrier containing the nucleic acid molecule described in n1); n4) A carrier containing the expression box described in n2); n5) A cell containing the nucleic acid molecules described in n1); n6) Cells containing the expression cassette described in n2); n7) Cells containing the carrier described in n3); n8) contains cells containing the carrier described in n4); n9) A cell comprising the RSV pre-fusion F protein mutant of any one of claims 1-3, the fusion protein of any one of claims 5-6, or the recombinant protein of claim 7; None of the cells described in n5)-n9) contain reproductive material.

9. A conjugate 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, or the recombinant protein according to claim 7; And a coupling portion, wherein the coupling portion is a detectable marker; 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 one of the applications (1)-(2); (1) The use of the RSV pre-fusion F protein mutant according to any one of claims 1-3, the complex according to claim 4, the fusion protein according to any one of claims 5-6, the recombinant protein according to claim 7, or the biomaterial according to claim 8 in any one of m1)-m2); m1) Prepare RSV antibody; m2) to prepare drugs; The drug is used for 11) or 12): l1) Prevent RSV infection; l2) Prevention and / or treatment of diseases caused by RSV infection; (2) The use of 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, the biomaterial according to claim 8, or the conjugate according to claim 9 in the preparation of the kit; The kit is used for any one of o1)-o3): o1) Detect the presence or content of RSV antibody in the sample; o2) Diagnosis of RSV infection; o3) Diagnose diseases caused by RSV infection; Preferably, the diseases caused by RSV infection include 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 of any one of claims 1-3, the complex of claim 4, the fusion protein of any one of claims 5-6, the recombinant protein of claim 7, or the biological material of claim 8.

13. The medicament according to claim 12, characterized in that, The drug also includes: a pharmaceutically acceptable carrier; Preferably, the drug further comprises: other active ingredients for the prevention and / or treatment of 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 1-3, the complex according to claim 4, the fusion protein according to any one of claims 5-6, the recombinant protein according to claim 7, or the biological material according to claim 8; And adjuvants.

15. The method for preparing 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, and the recombinant protein according to claim 7, wherein the protein is obtained by culturing the cells described in claim 8.

16. A method for preventing RSV infection or disease caused by RSV infection, comprising administering to a subject an effective amount of the drug of any one of claims 12-13 or the vaccine of claim 14.

17. A method comprising the steps of using the kit of claim 11; The method is used for any one of o1)-o3): o1) Detect the presence or content of RSV antibody in the sample; o2) Diagnosis of RSV infection; o3) Diagnose diseases caused by RSV infection.

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