RSV pre-fusion f protein mutants and uses thereof

Abstract

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

Description

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:

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

[0019] Compared with SEQ ID NO:3, the RSV pre-fusion F protein mutant contains at least one of the mutations in (1)-(3) (the resulting mutant is called the respiratory syncytial virus (RSV) A subtype pre-fusion F protein mutant):

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

[0021] 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 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 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;

[0023] 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.

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

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

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

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

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

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

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

[0031] In some embodiments, 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) 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.

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

[0033] In some embodiments, the RSV pre-fusion F protein mutant, compared to SEQ ID NO:3, 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).

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

[0035] In some embodiments, the RSV pre-fusion F protein mutant is associated with SEQ ID NO. Compared to NO:3, it includes any one of the mutations from f1) to 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 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] 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 including any one of the mutations f7), f14), f23), f25), f26), f47), f49), f54), f60), f78).

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

[0038] And the vector components that can display the mutant.

[0039] In some embodiments, the carrier component comprises at least one of nanomaterials, bacterial outer membrane vesicles (OMVs), polymerized pedestals, and virus-like particles (VLPs).

[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 derived from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

[0042] 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;

[0043] And trimerized base.

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

[0045] 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.

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

[0047] And optional tag sequences to assist in 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, 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.

[0050] A fifth aspect of the invention provides biomaterials related 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 biomaterials comprising any one of n1)-n9):

[0051] n1) A nucleic acid molecule encoding 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;

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

[0053] n3) A carrier containing the nucleic acid molecule described in n1);

[0054] n4) A carrier containing the expression box described in n2);

[0055] n5) A cell containing the nucleic acid molecules described in n1);

[0056] n6) Cells containing the expression cassette described in n2);

[0057] n7) Cells containing the carrier described in n3);

[0058] n8) contains cells containing the carrier described in n4);

[0059] n9) A cell 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.

[0060] In some embodiments, any of the cells described in n5)-n9) does not contain reproductive material.

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

[0062] In some embodiments, the eukaryotic expression vector includes yeast expression vectors, mammalian expression vectors, insect expression vectors, etc.

[0063] In some embodiments, any one of the cells (n5)-n9) is selected from prokaryotic cells and eukaryotic cells.

[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, cattle, pigs, dogs, and cats.

[0067] A 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 portion, wherein the coupling portion is a detectable marker.

[0069] 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.

[0070] 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.

[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, 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) Prepare RSV antibody;

[0074] m2) to prepare drugs;

[0075] The drug is used for l1) or l2):

[0076] l1) Prevent RSV infection;

[0077] l2) Prevention and / or treatment of diseases caused by RSV infection.

[0078] 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;

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

[0080] o1) Detect the presence or content of RSV antibody in the sample;

[0081] o2) Diagnosis of RSV infection;

[0082] o3) Diagnose diseases caused by RSV infection.

[0083] In some implementations, the RSV described in o1)-o3) includes the RSV A subtype.

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

[0085] 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.

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

[0087] o1) Detect the presence or content of RSV antibody in the sample;

[0088] o2) Diagnosis of RSV infection;

[0089] o3) Diagnose diseases caused by RSV infection.

[0090] In some implementations, the RSV described in o1)-o3) includes the RSV A subtype.

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

[0092] 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.

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

[0094] 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.

[0095] In some embodiments, the drug is used for l1) or l2):

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

[0097] l2) Prevention and / or treatment of diseases caused by RSV infection.

[0098] In some implementations, the RSV includes the RSV A subtype.

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

[0100] 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.

[0101] And adjuvants.

[0102] In some embodiments, the vaccine is used for l1) or l2):

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

[0104] l2) Prevention and / or treatment of diseases caused by RSV infection.

[0105] In some implementations, the RSV described in l1)-l2) includes the RSV A subtype.

[0106] In some embodiments, the diseases caused by the RSV infection include 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, obtained by culturing the cells of the fifth aspect of the present invention.

[0108] 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.

[0109] In some implementations, the RSV includes the RSV A subtype.

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

[0111] In some implementations, the subject may include mammals, such as humans or non-human mammals.

[0112] 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.

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

[0114] The method is applicable to any one of o1)-o3):

[0115] o1) Detect the presence or content of RSV antibody in the sample;

[0116] o2) Diagnosis of RSV infection;

[0117] o3) Diagnose diseases caused by RSV infection.

[0118] In some implementations, the RSV described in o1)-o3) includes the RSV A subtype.

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

[0120] The beneficial effects of this invention are:

[0121] This invention provides a pre-fusion F protein mutant of respiratory syncytial virus (RSV) with good stability, high expression level and / or immunogenicity. It 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

[0122] Figure 1 The neutralizing titer NT50 of RSV in the serum of mice immunized with the mutant was shown. Detailed Implementation

[0123] The present invention will be further described in detail below through specific embodiments.

[0124] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0125] 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.

[0126] Example 1

[0127] 1. Construction of plasmid expressing RSV pre-fusion F protein mutant (RSV A subtype pre-fusion 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 the 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 are used as examples:

[0131] (1) DS-CAV construction: ① In the first round of PCR, WT-F plasmid was used as a template. Short PCR fragments were amplified using primers (S155C-F; S290C-R), and long fragments were 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 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 (S190F-F; V207L-R), and long fragments were 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 shown in Table 3 for homologous recombination. (2) Construction of hd1b-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 PCR fragments were amplified using primers (V207L-R; E487C, A490C-F). 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; E487C, A490C-R), and long PCR fragments were amplified using primers (N228K-R; E487C, A490C-F). 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. (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, and other reagents are shown in Table 1. The PCR products were used in the reaction system shown 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; 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. (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 used for homologous recombination using the reaction system in Table 3.(6) Construction of hd4-2: ① In the first round of PCR, WT-F plasmid was used as a template. Short PCR fragments were amplified using primers (S55C-F; L188C-R), and long fragments were amplified using primers (S55C-R; L188C-F). 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 a template. Short PCR fragments were amplified using primers (V207L-RF; E487C, A490C-R), and long fragments were amplified using primers (V207L-RR; E487C, A490C-F). 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. (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, 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, WT-F plasmid was used 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, 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; 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. (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 used in the reaction system in Table 3 for homologous recombination. (10) Construction of he3b-3: Using plasmid (8)① 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 homologously recombinated using the reaction system in Table 3.(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 plasmid he4-1 as a template, the short PCR fragment primers were (N228K-F; Q501R-R), and the long fragment primers 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) hf3-3 construction: ① In the first round of PCR, WT-F plasmid was used as a template. Short PCR fragments were amplified using primers (V207L-F; L334I-R), and long fragments were 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 a template. Short PCR fragments were amplified using primers (S180C, S186C-F; E487C, A490C-R), and long fragments were 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 hf3-3 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, and other reagents are shown in Table 1. The PCR products were used in the reaction system shown 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 used in the reaction system shown in Table 3 for homologous recombination. (16) hf4b-1 construction: ① In the first round, (13)① plasmid 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 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 (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 The first round used WT-F plasmid as a template. 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 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; 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 used in the reaction system in Table 3 for homologous recombination.

[0132] Table 1 PCR reaction system

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

[0134] Table 2 Primer sequences

[0135]

[0136]

[0137] Table 3. Homologous recombination reaction system and conditions

[0138]

[0139] 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.

[0140] Table 4. Mutation sites corresponding to each mutant.

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] 2. Detecting the stability of mutants

[0147] The stability of the mutant was assessed as follows:

[0148] 1) Use a 96-well plate with 1x10 holes per well. 4 100 μL of 293t cells were seeded into plates, and on the second day, the plasmid of the RSV pre-fusion F protein mutant 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.

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

[0150] 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.

[0151] 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).

[0152] Table 5. Results of single mutations in disulfide bonds (5-fold dilution)

[0153]

[0154] Note: "D25" indicates that the D25 antibody test was used.

[0155] Table 6. Results of disulfide bond double mutagenesis (5-fold dilution)

[0156]

[0157]

[0158] Note: "D25" indicates that the D25 antibody test was used.

[0159] Table 7. Results of electrostatic single mutations (5-fold dilution)

[0160]

[0161]

[0162] Note: "D25" indicates that the D25 antibody test was used.

[0163] Table 8. Results of single mutations in the cavity (undiluted)

[0164]

[0165]

[0166] Note: "D25" indicates that the D25 antibody test was used.

[0167] Table 9. Results of electrostatic / cavitary double mutation (5-fold dilution)

[0168]

[0169] Note: "D25" indicates that the D25 antibody test was used.

[0170] Table 10 Results of three types of combined mutations (n=3) (125-fold dilution)

[0171]

[0172]

[0173]

[0174]

[0175]

[0176] Note: "D25" indicates that D25 antibody detection was used, and "AM14" indicates that AM14 antibody detection was used.

[0177] 3. RSV-F and related neutralizing antibody affinity assay

[0178] 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:

[0179] 1) The cell density reached 2x10 at 293f. 6 / 100μL, transfected with PEI (transfection plasmid amount is 2μg / mL);

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

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

[0182] 4) 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) was tested using a Biacore 8K surface plasmon resonance (SPR) instrument (refer to the instruction manual for the detection method).

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

[0184] Table 11 Results of antigen epitope affinity

[0185]

[0186]

[0187] 4. Test expression levels

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

[0189] 1) Detect the expression levels of some mutants, specifically as follows: using a 96-well plate with 1x10 μL per well. 4 100 μL of 293t cells were seeded into plates, and the plasmid was transfected with PEI (100 ng / well) on the second 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 by sandwich ELISA (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, 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 comparable to DS-CAV.

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

[0191]

[0192]

[0193] Note: "D25" indicates that the D25 antibody test was used.

[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) The cell density reached 2x10 at 293f. 6 / 100μL, transfected with PEI (transfection plasmid amount is 2μg / mL);

[0196] 2) Six 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, and cell supernatant was obtained.

[0197] 3) Use vacuum filtration to filter the supernatant again to prevent clogging of the purification column;

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

[0199] 5) Concentrate the eluted protein solution using a concentration tube and replace the buffer with 1XPBS;

[0200] 6) Use the BCA protein assay method to determine the protein concentration;

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

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

[0203] 6. Mouse immunization

[0204] Five Balb / c mice were immunized with each protein antigen. 50 μL of the protein solution obtained in step 3 above (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.

[0205] 7. Neutralization Experiment

[0206] 1) Use culture medium containing 2% fetal bovine serum at a ratio of 1 x 10⁻⁶ cells / well. 4 / 100μL of hep2 cells were plated;

[0207] 2) The mouse serum obtained in step 6 was uniformly diluted 10-fold, and then serially diluted 3-fold (the diluent was 100 TCID50 RSV virus solution) (GenBank: MW582527.1), for a total of 7 dilutions. After mixing, the mixture was incubated at 37°C for 1 hour, with the mixture being inverted and shaken once during the incubation period.

[0208] 3) Add 100 μL of diluted serum from 2) to the well plate in 1), with 6 replicates per well for each dilution gradient. At the same time, set up positive control (RSV virus solution with 400 TCID50) and negative control (culture medium containing 2% fetal bovine serum).

[0209] 4) After two days of incubation, remove the supernatant twice with PBS detergent, add 4% paraformaldehyde, and seal with sealant.

[0210] 5) Using high-content scanning plates, the fluorescence area of ​​each well was counted, and neutralization data analysis was performed;

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

[0212] The results are as follows Figure 1 As shown, 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 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 RSV pre-fusion F protein mutant, compared with SEQ ID NO:3, has the following mutation site: S180C, S186C, E487C, A490C, V207L, N228K and Q501R; or S180C, S186C, E487C, A490C, and N228K; or S180C, S186C, E487C, A490C, N228K and Q501R; or S180C, S186C, E487C, A490C, V207L ​​and L334I; or S180C, S186C, E487C, A490C, V207L ​​and N228K; or S180C, S186C, E487C, A490C, V296I, and Q501R; or S180C, S186C, E487C, A490C, V296I, and N228K; or S180C, S186C, E487C, A490C, V207L, L334I, and Q501R; or S180C, S186C, E487C, A490C, V207L ​​and Q501R; or S180C, S186C, E487C, A490C, V296I, L334I, and Q501R; or S180C, S186C, E487C, A490C, V296I, N228K and Q501R; or S180C, S186C, E487C, A490C, V207L, L334I, N228K and Q501R; or S180C, S186C, E487C, A490C, V296I, L334I and N228K.

2. A complex comprising the RSV pre-fusion F protein mutant of claim 1; And the vector components that can display the mutant.

3. The complex according to claim 2, characterized in that, The carrier component comprises at least one of nanomaterials, bacterial outer membrane vesicles (OMVs), polymerized substrates, and virus-like particles (VLPs).

4. A fusion protein comprising, from the N-terminus to the C-terminus, the RSV pre-fusion F protein mutant of claim 1 and a trimerizing base, wherein the amino acid sequence of the trimerizing base is shown in SEQ ID NO:

4.

5. A recombinant protein comprising the fusion protein of claim 4 and optionally a tag sequence for expression and / or purification.

6. The recombinant protein according to claim 5, characterized in that, 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.

7. A biomaterial relating to the RSV pre-fusion F protein mutant of claim 1, the fusion protein of claim 4, or the recombinant protein of any one of claims 5-6, wherein the biomaterial comprises any one of n1)-n9): n1) A nucleic acid molecule encoding the RSV pre-fusion F protein mutant of claim 1, the fusion protein of claim 4, or the recombinant protein of any one of claims 5-6; n2) 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) Cells containing the carrier described in n4); n9) Cells comprising the RSV pre-fusion F protein mutant of claim 1, the fusion protein of claim 4, or the recombinant protein of any one of claims 5-6; None of the cells described in n5)-n9) contain reproductive material.

8. A conjugate comprising the RSV pre-fusion F protein mutant of claim 1, the fusion protein of claim 4, or the recombinant protein of any one of claims 5-6; And a coupling portion, wherein the coupling portion is a detectable marker.

9. The conjugate of claim 8, wherein, The detectable markers include at least one of luminescent markers, electron-dense markers, radioactive markers, and enzyme markers.

10. The conjugate of claim 8, wherein, The detectable markers include at least one of metal particles, fluorescent markers, and chemiluminescent markers.

11. Any one of (1) or (2); (1) The use of the RSV pre-fusion F protein mutant of claim 1, the complex of any one of claims 2-3, the fusion protein of claim 4, the recombinant protein of any one of claims 5-6, or the biomaterial of claim 7 in any one of m1)-m2); m1) Prepare RSV antibody; m2) to prepare drugs; The drug is used for l1) or l2): l1) Prevent RSV infection; l2) Prevention of diseases caused by RSV infection; (2) The use of the RSV pre-fusion F protein mutant of claim 1, the fusion protein of claim 4, the recombinant protein of any one of claims 5-6, the biomaterial of claim 7, or the conjugate of any one of claims 8-10 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.

12. Use according to claim 11, characterized in that, The diseases caused by RSV infection include at least one of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

13. A kit comprising: the RSV pre-fusion F protein mutant of claim 1, the fusion protein of claim 4, the recombinant protein of any one of claims 5-6, or the conjugate of any one of claims 8-10.

14. A drug comprising: the RSV pre-fusion F protein mutant of claim 1, the complex of any one of claims 2-3, the fusion protein of claim 4, the recombinant protein of any one of claims 5-6, or the biological material of claim 7.

15. The medicament according to claim 14, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

16. The medicament according to any one of claims 14-15, characterized in that, The drug also contains other active ingredients for the prevention and / or treatment of RSV infection or diseases caused by RSV infection.

17. A vaccine comprising: the RSV pre-fusion F protein mutant of claim 1, the complex of any one of claims 2-3, the fusion protein of claim 4, the recombinant protein of any one of claims 5-6, or the biological material of claim 7; And adjuvants.

18. The method for preparing the RSV pre-fusion F protein mutant of claim 1, the fusion protein of claim 4, and the recombinant protein of any one of claims 5-6, obtained by culturing the cells of claim 7.

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