CN111978400B

CN111978400B - Rift valley fever virus humanized monoclonal antibody and application thereof

Info

Publication number: CN111978400B
Application number: CN202010860411.3A
Authority: CN
Inventors: 严景华; 王奇慧; 马桐; 杨化冰; 高福; 马素芳
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2019-02-15
Filing date: 2019-02-15
Publication date: 2022-04-01
Anticipated expiration: 2039-02-15
Also published as: CN111978400A; CN114409770B; CN109734800B; CN114409770A; CN114409769B; CN109734800A; CN114409769A

Abstract

The invention discloses a rift valley fever virus humanized monoclonal antibody and application thereof, belonging to the technical field of medicines. The invention takes RVFV glycoproteins Gn and Gc expressed by baculovirus as antigens, memory B cells capable of combining the RVFV glycoproteins Gn and Gc are screened from PBMCs of a convalescent patient with rift valley fever, and 8 Gn human monoclonal antibodies and a Gc human monoclonal antibody which are efficiently neutralized by the RVFV infection are identified through single B cell sequencing, in vitro neutralization, in vivo protection and other experiments. The human monoclonal antibodies of the invention have very high in vitro neutralizing activity (IC)₅₀Can be as low as 1.93 plus or minus 0.6pM and reaches pM level), can effectively treat mice infected by RVFV, prevent the infection of the RVFV to the mice, and has extremely high clinical treatment and RVFV infection prevention application values.

Description

Rift valley fever virus humanized monoclonal antibody and application thereof

Technical Field

The invention relates to a rift valley fever virus humanized monoclonal antibody and application thereof, belonging to the technical field of medicines.

Background

Rift Valley Fever Virus (RVFV) is one of the insect-borne viruses that seriously threaten human and animal health, belongs to the family of bunyavirales (bunyavirales) parvoviridae (phenouiviridae) genus Phlebovirus (Phlebovirus), and causes fever in Rift Valley (RVF) which is a common disease of humans and animals, resulting in death and abortion in livestock. The surface of the virus is covered with an envelope and glycoprotein protrusion, and a virus genome (S, M and L) is formed by 3 segments of negative strand RNA, wherein, the M segment encodes surface glycoproteins Gn and Gc, and encodes a non-structural protein (NSm) at the 5' end of Gn, and Gn and Gc are main envelope proteins of the virus and are key proteins for the invasion and the membrane fusion of the virus; the spread range is very wide, and sheep, goats, cattle, buffalo, camel and other livestock can be infected with the virus; the mortality rate is very high, especially for young animals, once infected, the mortality rate approaches 100%.

Humans are also infected with RVFV and most of the symptoms are mild and self-healing, but some patients develop severe symptoms, of which 5-10% of the patients' retinas produce degenerative changes and severe symptoms can lead to permanent blindness in one or both eyes; hemorrhagic fever occurs in < 1% of patients, of which 50% die; encephalitis appears in < 1% of patients, and patients with the symptoms have low mortality rate, but usually have serious sequelae such as hemiplegia and the like.

In 1930, scientists Danbney et al first isolated RVFV in a single sheep disease outbreak survey of large grains in kenya, and since this, found that RVF epidemics were regularly outbreaks in animals and humans, causing significant losses. For example, in 1977-1978, the RVF epidemic was outbreak in Egypt, when a total of 20 ten thousand infections were present, including 18,000 severe patients and 600 deaths; from 2000 to 2018, the World Health Organization (WHO) was globally informed of 4830 severe RVF cases, including 967 deaths, with a mortality rate of about 20.0%.

Geographically, RVFV is prevalent in african regions in general. However, in 9 months of 2000, the arabian peninsula (saudi arabia and also africa) also developed an RVF epidemic, which was also reported in the first areas outside africa; in 2016, a single case of input occurred in our country, and the patient returned to China from angora.

In terms of mode of transmission, RVFV is transmitted primarily by mosquito bites. As many as 30 species of mosquitoes are known to transmit the disease, distributed on various continents other than the arctic, and therefore, the risk of the RVFV continuing to spread to the world is very high.

Currently, an effective prophylactic against RVFV infection in animals is a vaccine. In the field of veterinary vaccines, attenuated live vaccines of RVFV are used in Kenya and south Africa, and inactivated formalin vaccines are used in Egyptian and south Africa. Generally, attenuated live vaccines are only effective in sheep immunization, cannot protect cattle, have high side effects, and often cause abortion in ewes; although the formalin inactivated vaccine has higher safety, the formalin inactivated vaccine has the problems of high cost and non-durable immunity. While other types of vaccines are still under investigation, including: DNA vaccines, genetically engineered strains, VLPs, and the like.

Effective prevention of RVFV infection in humans remains mainly by immunizing livestock, cutting off the source of infection. In addition, high risk groups in african regions can be vaccinated with formalin inactivated vaccines, but due to cost issues, extensive vaccination is not possible. After human disease, ribavirin (ribavirin) may have therapeutic effects, but the current clinical data are few.

Therefore, there is a need to develop an effective therapeutic agent for RVFV infection, particularly a treatment that can cure critically ill patients. In this regard, antibodies show great potential.

Disclosure of Invention

In order to solve the problems, the invention researches on RVFV vaccine, and finds that Gn alone or Gn and Gc together in vaccine form can effectively generate RVFV neutralizing antibody, that is, the antibody against Gn or Gc is very likely to neutralize RVFV infection, and becomes a medicament for treating RVFV infection.

Therefore, the invention selects RVFV glycoproteins Gn and Gc expressed by baculovirus as antigens from PBMCs of a convalescent patient with a cracked valley fever, and identifies 8 Gn human monoclonal antibodies (R4, R12, R13, R15, R16, R17, R19, R22) and a Gc human monoclonal antibody (R5) which can effectively neutralize RVFV infection through single B cell sequencing, in vitro neutralization and in vivo protection.

The human monoclonal antibodies of the invention have very high in vitro neutralizing activity (IC)₅₀Can be as low as 1.93 plus or minus 0.6pM and reaches pM level), can effectively treat mice infected by RVFV, prevent the infection of the RVFV to the mice, and has extremely high clinical treatment and RVFV infection prevention application values.

The technical scheme of the invention is as follows:

the present invention provides an antibody or antigen-binding fragment comprising a heavy chain and a light chain; the heavy chain comprises a heavy chain variable region and a heavy chain constant region; the light chain comprises a light chain variable region and a light chain constant region; the antibody or antigen binding fragment is named as R4, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2;

or the antibody or the antigen binding fragment is named as R12, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4;

or the antibody or the antigen-binding fragment is named as R13, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 6;

or the antibody or the antigen-binding fragment is named as R15, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8;

or the antibody or the antigen-binding fragment is named as R16, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 9, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10;

or the antibody or the antigen-binding fragment is named as R17, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 11, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 12;

or the antibody or the antigen-binding fragment is named as R19, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 13, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 14;

or the antibody or antigen binding fragment is named as R22, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 15, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 16;

or the antibody or the antigen-binding fragment is named as R5, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 17, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 18.

In one embodiment of the invention, the amino acid sequences of the heavy chain constant regions of R4, R12, R13, R15, R16, R17, R19, R22 and R5 are shown as SEQ ID NO 19.

In one embodiment of the invention, the amino acid sequences of the light chain constant regions of R4, R12, R13, R15, R16, R17 and R5 are shown as SEQ ID NO 20; the amino acid sequences of the light chain constant regions of R19 and R22 are shown as SEQ ID NO 21.

In one embodiment of the invention, the heavy chain and the light chain of R4, R12, R13, R15, R16, R17 and R5 are all connected through EcoR I and Xho I; the heavy chains of R19 and R22 are connected by EcoR I and Xho I, and the light chains are connected by SacI and Xho I.

The invention provides a nucleotide sequence encoding the above antibody or antigen-binding fragment, said nucleotide sequence comprising a nucleotide sequence encoding a heavy chain and a nucleotide sequence encoding a light chain; the nucleotide sequence of the coding heavy chain sequentially comprises a CMV promoter sequence, a leader sequence, a sequence for coding a heavy chain variable region and a sequence for coding a heavy chain constant region;

or the nucleotide sequence of the coding heavy chain sequentially comprises a CMV promoter sequence, a connecting sequence, a leader sequence, a sequence for coding a heavy chain variable region, a sequence for coding a heavy chain constant region and a connecting sequence.

In one embodiment of the invention, when the heavy chain-encoding nucleotide sequence does not comprise a linker sequence, the heavy chain-encoding nucleotide sequences may be linked by PCR.

In one embodiment of the present invention, when the nucleotide sequence encoding the heavy chain comprises a linker sequence, the linker sequence may be a sequence of enzyme cleavage sites.

In one embodiment of the present invention, when the nucleotide sequence encoding the heavy chain comprises a linker sequence, the nucleotide sequence encoding the heavy chain comprises a CMV promoter sequence, an EcoR I cleavage site sequence, a leader sequence, a sequence encoding the heavy chain variable region, a sequence encoding the heavy chain constant region, and an Xho I cleavage site sequence in this order.

In one embodiment of the invention, the nucleotide sequence encoding the light chain comprises, in order, a CMV promoter sequence, a leader sequence, a sequence encoding the variable region of the light chain, a sequence encoding the constant region of the light chain;

or the nucleotide sequence for coding the light chain sequentially comprises a CMV promoter sequence, a connecting sequence, a leader sequence, a sequence for coding a light chain variable region, a sequence for coding a light chain constant region and a connecting sequence.

In one embodiment of the invention, the nucleotide sequences encoding the heavy chain may be linked by PCR when the nucleotide sequences encoding the light chain do not comprise a linking sequence.

In one embodiment of the present invention, when the nucleotide sequence encoding the light chain comprises a linker sequence, the linker sequence may be a sequence of cleavage sites.

In one embodiment of the present invention, when the nucleotide sequence encoding a light chain comprises a linker sequence, the nucleotide sequence encoding a light chain is divided into a nucleotide sequence encoding R4, R12, R13, R15, R16, R17, R19, and R22 light chains and a nucleotide sequence encoding R5 light chains; the nucleotide sequences of the codes R4, R12, R13, R15, R16, R17, R19 and R22 light chains sequentially comprise a CMV promoter sequence, a Sac I enzyme cutting site sequence, a leader sequence, a sequence for coding a light chain variable region, a sequence for coding a light chain constant region and an Xho I enzyme cutting site sequence; the nucleotide sequence of the coding R5 light chain comprises a CMV promoter sequence, an EcoR I enzyme cutting site sequence, a leader sequence, a sequence of a coding light chain variable region, a sequence of a coding light chain constant region and an Xho I enzyme cutting site sequence in sequence.

In one embodiment of the invention, the amino acid sequence of the leader sequence is set forth in SEQ ID NO 22.

The present invention provides a plasmid containing the above nucleotide sequence.

In one embodiment of the invention, the plasmid is a viral plasmid.

The present invention provides a host cell containing the above plasmid.

The invention provides the use of an antibody or antigen-binding fragment as defined above or a nucleotide sequence as defined above or a plasmid as defined above or a host cell as defined above for the preparation of a medicament for the treatment and/or prophylaxis of rift Valley fever virus.

The invention provides a pharmaceutical composition comprising a prophylactically effective amount of the above-described antibody or antigen-binding fragment.

In one embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

The invention provides a kit comprising the above antibody or antigen-binding fragment or the above nucleotide sequence or the above plasmid or the above host cell.

Has the advantages that:

(1) the invention obtains 8 strains of human source high-neutralization active antibodies which are combined with Gn by screening memory B cells which are specifically combined with Gn and Gc of RVF rehabilitation patients: r4, R12, R13, R15, R16, R17, R19 and R22, and 1 strain Gc-binding human neutralizing antibody: r5;

(2) the 8 strain Gn-specific antibodies of the invention have very high in vitro neutralization activity, wherein IC of two strain Gn-specific antibodies R15 and R16₅₀Can reach pM grade, and the in vivo detection result shows that the 8 Gn-specific antibodies can effectively treat mice infected by the RVFV and prevent the mice from being infected by the RVFV; meanwhile, the 1-strain Gc-specific antibody R5 can neutralize RVFV infection at high dose;

(3) the 8 strain Gn-specific humanized antibody and the 1 strain Gc-specific humanized antibody have extremely high application value in the aspects of clinical treatment and RVFV infection prevention.

Drawings

FIG. 1: RVFVn protein purification molecular sieve and SDS-PAGE result.

FIG. 2: RVFV Gc protein purification molecular sieve and SDS-PAGE result.

FIG. 3: the results of the alignment of the R12 light chain and R13 light chain with germline genes.

FIG. 4: the results of the alignment of the R16 light chain and R17 light chain with germline genes.

FIG. 5: molecular sieve chromatography results of R4 purification.

FIG. 6: molecular sieve chromatography results of R5 purification.

FIG. 7: molecular sieve chromatography results of R12 purification.

FIG. 8: molecular sieve chromatography results of R13 purification.

FIG. 9: molecular sieve chromatography results of R15 purification.

FIG. 10: molecular sieve chromatography results of R16 purification.

FIG. 11: molecular sieve chromatography results of R17 purification.

FIG. 12: molecular sieve chromatography results of R19 purification.

FIG. 13: molecular sieve chromatography results of R22 purification.

FIG. 14: kinetic profiles of antibody binding to RVFV Gn and Gc.

FIG. 15: neutralization profile of antibody to RVFV.

FIG. 16: therapeutic effect of antibodies on RVFV infected mice.

FIG. 17: the effect of the antibody in preventing mice from being infected with RVFV.

FIG. 18: blocking effect of RVFV antibodies on Gn tetramer binding to Huh7 cells.

FIG. 19: the blocking effect of the RVFV antibody on the binding of RVFV virions to Vero cells.

Detailed Description

The invention is further illustrated with reference to specific examples.

The pFastBac1 vector and pCAGGS vector referred to in the following examples were purchased from Youbao organisms; coli DH10Bac competent cells, HEK293T cells, and Hi5 insect cells referred to in the examples below were purchased from Saimer Feishell science and technology (China) Co., Ltd.; insect SF9 cells referred to in the examples below were purchased from North Naphthora.

Example 1: expression and purification of RVFV Gn and Gc proteins

Genes for coding RVFVn (amino acid sequence is shown as SEQ ID NO: 23) and Gc (amino acid sequence is shown as SEQ ID NO: 24) proteins are respectively connected into a pFastBac1 vector, wherein, the N ends of the Gn and Gc protein genes are connected with an insect cell membrane protein Gp67 signal peptide sequence for secretion of antibodies; the end C is connected with a His tag, so that purification and B cell staining are facilitated; all encoding genes were codon optimized for insect cells.

Coli DH10Bac competent cells were transformed with pFastBac1 vector containing the target gene, and recombinant baculovirus was screened by Blue-white spot on a plate containing ampicillin, kanamycin, tetracyclin and Blue-gal. After the white bacterial plaque is identified by PCR of the upstream primer and the downstream primer of M13, the bacterial strain is cloned positively, and the recombinant Bacmid is extracted by an isopropanol precipitation method.

Insect SF9 cells were transfected with recombinant Bacmid and cultured in serum-free medium for 3 days to observe changes in cell morphology and proliferation. When SF9 cells were significantly swollen, few dead cells appeared, and the viral fluid supernatant was harvested without proliferation, this was passage P1. After continued baculovirus expansion to P4 passages, Hi5 insect cells were infected for Gn and Gc expression.

Hi5 cell culture supernatant containing the target protein was centrifuged and filtered (0.22. mu.M) to remove cell debris, and then bound to a HisTrap FF (GE healthcare) nickel chelate column, and then eluted with imidazole-eluting chromatography columns of different concentrations to collect the eluted proteins, respectively, and the samples containing the target protein were judged from the SDS-PAGE results.

Collecting the elution peak containing the target protein, concentrating, performing molecular sieve chromatography, determining the peak position according to the protein size, and determining the size and purity of the protein according to the result of SDS-PAGE (the SDS-PAGE and molecular sieve results are shown in figure 1-2).

Example 2: isolation of RVFVn from Gc protein specific memory B cells

With patient informed consent, 30mL of blood was collected and PBMCs were isolated.

Isolating the PBMCs at 10⁷The density of individual/mL was combined with final concentrations of 100nM RVFV Gn and RVFV Gc proteins incubated on ice for half an hour, then washed 2 times with PBS and incubated on ice for half an hour with the following antibodies: anti-human CD3/PE-Cy5, anti-human CD16/PE-Cy5, anti-human CD235a/PE-Cy5, anti-human CD19/APC-Cy7, anti-human CD27/Pacific Blue, anti-human CD38/APC, anti-human IgG/FITC, and anti-His/PE, followed by washing 2 times with PBS.

PE-Cy5-APC-APC-Cy7+ Pacific Blue + FITC + PE + cells were collected by FACSAria III sorting and directly collected in a 96-well plate at 1 cell/well.

Example 3: single B cell PCR and sequence analysis

The cells obtained in example 2 were reverse-transcribed by Superscript III reverse transcriptase (Invitrogen) and reacted at 55 ℃ for 60 min.

Using this reverse transcription product as a template, PCR was performed using HotStar Tap Plus enzyme (QIAgen) to amplify antibody variable region sequence (PCRa) under the following reaction conditions: 95 ℃ for 5 min; 95 ℃, 30s, 55 ℃ (heavy chain/kappa chain)/50 ℃ (lambda chain), 30s, 72 ℃, 90s, 35 cycles, 72 ℃, 7 min.

This was used as a template for 1 additional round of PCR (PCRb) under the following conditions: 95 ℃ for 5 min; 95 ℃, 30s, 58 ℃ (heavy chain)/60 ℃ (kappa chain)/64 ℃ (lambda chain), 30s, 72 ℃, 90s, 35 cycles, 72 ℃, 7 min.

1.2% agarose gel electrophoresis, separating PCR products, recovering the cut gel with the size of 400-500bp, sending the cut gel to a sequencing company for sequencing, and analyzing the sequencing result by using IMGT online software.

Analyzing the correct variable region sequence, connecting with the constant region of the corresponding heavy chain/kappa chain/lambda chain through bridging PCR, cloning into an expression vector pCAGGS, and obtaining a recombinant plasmid containing the specific antibody light and heavy chain coding genes; wherein the heavy chain is connected with the lambda chain by EcoR I and Xho I, and the kappa chain is connected with the Xho I by Sac I.

The human antibody design strategy is as follows:

heavy chain: CMVpromoter-EcoR I-Leader sequences-heavy chain variable region-C_H-Xho I；

Light chain (κ): CMVpromoter-Sac I-Leader sequences-light chain variable region-C_L(κ)-Xho I；

Light chain (λ): CMVpromoter-EcoR I-Leader sequences-light chain variable region-C_L(λ)-Xho I；

9 antibodies were obtained: r4, R12, R13, R15, R16, R17, R19, R22 and R5, wherein the amino acid sequence of the heavy chain variable region of R4 is shown as SEQ ID NO. 1 and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the heavy chain variable region of R12 is shown in SEQ ID NO. 3 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 4; the amino acid sequence of the heavy chain variable region of R13 is shown in SEQ ID NO. 5 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 6; the amino acid sequence of the heavy chain variable region of R15 is shown in SEQ ID NO. 7 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 8; the amino acid sequence of the heavy chain variable region of R16 is shown in SEQ ID NO. 9 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 10; the amino acid sequence of the heavy chain variable region of R17 is shown in SEQ ID NO. 11 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 12; the amino acid sequence of the heavy chain variable region of R19 is shown in SEQ ID NO. 13 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 14; the amino acid sequence of the heavy chain variable region of R22 is shown in SEQ ID NO. 15 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16; the amino acid sequence of the heavy chain variable region of R5 is shown in SEQ ID NO. 17 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 18; c_HThe amino acid sequence of the region is shown as SEQ ID NO. 19; c_L(κ)The amino acid sequences of the regions (i.e., the light chains of R4, R12, R13, R15, R16, R17, and R5) are set forth in SEQ ID NO: 20; c_L(λ)The amino acid sequences of the regions (i.e., the light chain of R19 and R22) are set forth in SEQ ID NO: 21; the amino acid sequence of Leader sequences is shown in SEQ ID NO: 22.

The light chain sequences of R4, R12, R13, R15, R16, R17, R19, R22 and R5 were aligned with germline genes, respectively, wherein the alignment results of the light chain of R12 and the light chain of R13 and the germline genes are shown in fig. 3, and the alignment results of the light chain of R16 and the light chain of R17 and the germline genes are shown in fig. 4.

As shown in fig. 3, sequence analysis showed that the R12 light chain and R13 light chain had the same germline genes with the highest sequence identity, i.e. IGLV5-37 × 01 and LJ3 × 02, and that the R12 light chain and R13 light chain had the same CDR3 sequences; as shown in fig. 4, sequence analysis showed that R16 and R17 light chains had identical germline genes with the highest sequence identity, i.e., IGLV3-21 × 03 and LJ1 × 01, but the CDR3 sequences and numbers of R16 light chains and R17 light chains differed due to the different patterns of sequence V and J genes in sequence insertions and deletions.

Example 4: expression and purification of antibodies

Culturing 293T cells by DMEM containing 10% FBS, co-transfecting 293T cells by the recombinant plasmid containing the specific antibody light and heavy chain coding genes obtained in the example 3, changing the cell liquid into serum-free DMEM after 4-6 hours of transfection, continuously culturing for 3 days, collecting supernatant, supplementing DMEM, culturing for 4 days again, and collecting supernatant.

The collected supernatant was centrifuged at 5000rpm for 30min, mixed with an equal volume containing 20mM sodium phosphate (pH 8.0), filtered through a 0.22 μm filter, bound to a proteinA pre-packed column (5mL, GE Healthcare), and the bound protein was eluted with 10mM glycine (pH 3.0), collected and concentrated for molecular sieve chromatography, and the peaks were confirmed by SDS-PAGE to obtain purified antibodies R4, R12, R13, R15, R16, R17, R19, R22, and R5 (the results of molecular sieve chromatography are shown in FIGS. 5-13).

Example 5: detection of antibody Performance

(1) Detection of binding capacity of antibody and RVFV Gn and Gc by surface plasmon resonance technology

Surface plasmon resonance analysis was performed using Biacore T100(Biacore Inc.) with the following specific steps:

antibody of anti-human IgG is fixed on a channel (Fc) 1 and an Fc2 of a CM5 chip in an amino coupling mode, the fixed quantity is controlled to be about 10000 response values (RU), the channel is adjusted to Fc2, and then the purified antibody obtained in example 4 is combined in an antibody capture mode, wherein the liquid flow speed is controlled to be 10 mu L/min, the sample injection is carried out for 1min, and the antibody capture quantity is about 100 RU.

After diluting ZIKV-E protein in a 10mM HEPES, 150mM NaCl, pH 7.4 solution at a double ratio, adjusting the flow rate to 30. mu.L/min, and adjusting the channel to a pattern of Fc2-Fc1, RVFV Gn protein or Gc was loaded one by one from a low concentration, wherein the calculation of the binding kinetic constants was performed using BIAevaluation software T100(Biacore, Inc.) software (kinetic curves of antibody binding to Gn or Gc are shown in FIG. 14, and affinity of antibody to RVFV Gn and Gc is shown in Table 1).

TABLE 1 affinity of the antibodies for RVFV Gn and Gc

(2) Neutralization test

The purified antibody obtained in example 4 was diluted at a 3-fold ratio, mixed with 8 × 103PFU of RVFV (Vero expansion), incubated at 37 ℃ for 60 minutes, and then the mixture was added to a 24-well plate filled with Vero cells at 300 μ L/well, incubated at 37 ℃ for 1 hour, supplemented with 1mL of medium (DMEM, 10% FBS) per well, and cultured for another 48 hours and then stained.

The cells were collected, treated with 4% paraformaldehyde, 0.05% soponin in PBS, and left on ice in the dark for 30 min. Then, the cells were washed 2 times with a solution (PBS, 1% BSA, 0.01% solution), incubated with 2. mu.g/mL of R4 antibody on ice for 30min, then washed 2 times with a solution, incubated with 1:200 diluted anti-human IgG away from light on ice for 30min, washed 2 times with a solution, and then the positive ratio of the cells was measured by FACSCAnto, and the neutralizing capacity of the antibody against RVFV was calculated from the positive ratio at different concentrations (the neutralizing curve of the antibody against RVFV is shown in FIG. 15, and the neutralizing capacity of the antibody against RVFV in vitro is shown in Table 2).

TABLE 2 in vitro neutralizing Capacity of antibodies to RVFV

Ab	IC₅₀(Mean±SD，pM)
		R4	312±146
R5	21600±21600
		R12	12.3±10.7
R13	375±210
		R15	3.53±1.67
R16	1.93±0.6
		R17	16.9±15.9
R19	341±131
		R22	481±3.93

(3) Animal protection test

RVFV infection can cause BALB/c mice to die, and the effect of the purified antibody obtained in example 4 on RVFV infection is examined by using the same type of antibody as a negative control and using two methods of prevention and treatment.

The treatment effect is as follows: mice were divided into 4-5 groups, each mouse was intraperitoneally injected with 1x 103PFU of RVFV, 24 hours after infection, and then each mouse was intraperitoneally injected with a single dose of antibody at a dose of 10mg/kg, and survival and weight changes of the mice were recorded within 14 days, and mice with weight changes of more than 20% were sacrificed, wherein Z3L1 group was injected with the same type of antibody (results are shown in fig. 16).

The prevention effect is as follows: mice were divided into groups of 4-5, each mouse was intraperitoneally injected with a single dose of antibody of 10mg/kg, 24 hours later, mice were sacrificed by intraperitoneally injecting RVFV of 1x 103PFU, respectively, and survival and weight change of the mice were recorded within 14 days, with more than 20% of the mice being sacrificed, wherein Z3L1 group was injected with the same type of antibody (results are shown in fig. 17).

(4) Detection of antibody mechanism of action

A biotinylated tag was introduced at the C-terminus of Gn, expressed by HEK293T cells, and purified by HisTrap FF (GE healthcare) nickel chelate column and molecular sieve column chromatography. The purified Gn protein is reacted by BirA, and a Biotin molecule is added on a biotinylation label. The Biotin molecule has extremely high binding capacity with Streptavidin. After adding Streptavidin/PE to Gn-biotin Gn will label PE fluorescein and form tetramer. The 10. mu.g/mL Gn tetramer was incubated with 0.6mg/mL antibody, then bound to Huh7 cells, then to anti-hIgG/APC at a final concentration of 5. mu.g/mL, and finally subjected to flow analysis (the results are shown in FIG. 18).

5×10⁴PFU RVFV virus was incubated with 200. mu.g/mL antibody at 37 ℃ for 1 hour, cooled on ice, and pre-cooled to 2X 10⁵The Vero cells of (5) were incubated on ice for 30 min. After washing with PBS, the cells were fixed with 0.5% paraformaldehyde on ice for 30min and then bound to anti-hIgG/APC at a final concentration of 5. mu.g/mL (binding results are shown in FIG. 19).

In conclusion, 8 human source high-neutralizing active antibodies which are combined with Gn are obtained by screening memory B cells which are specifically combined with Gn and Gc of RVF rehabilitation patients: r4, R12, R13, R15, R16, R17, R19 and R22, and 1 strain Gc-binding human neutralizing antibody: and R5.

Although the binding constant of 8 Gn-specific antibodies to Gn is low (Table 1), the antibody has very high in vitro neutralizing activity (Table 2), wherein the IC50 of two Gn-specific antibodies, namely R15 and R16 reaches pM level, and the in vivo detection result shows that the 8 Gn-specific antibodies can effectively treat mice infected by RVFV (figure 16), and the 8 antibodies can also effectively prevent the infection of RVFV (figure 17), further experiments show that the Gn-specific antibody of RVFV can block the binding of Gn to susceptible cells (figure 18), and virus level experiments also reveal that the RVGn-specific antibody can block the binding of RVFV virion to susceptible cells (figure 19).

Therefore, the Gn-specific antibody functions to inhibit viral infection by binding to Gn on the RVFV virion and blocking binding of the virus to cells.

Although the neutralizing activity of Gc-specific antibody R5 was weak (table 2), RVFV infection was still neutralized at high doses.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> rift valley fever virus human monoclonal antibody and application thereof

<160> 24

<170> PatentIn version 3.3

<210> 1

<211> 116

<212> PRT

<213> Artificial sequence

<400> 1

Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Thr Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Asn Pro Thr Tyr Ala Gln Ala Phe

50 55 60

Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Trp Gly Trp Phe Asp Pro Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 2

<211> 116

<212> PRT

<213> Artificial sequence

<400> 2

Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ala Gly

20 25 30

Tyr Asp Val His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Arg

35 40 45

Leu Ile Tyr Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser Ser

85 90 95

Leu Ser Phe Ser Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

100 105 110

Gln Pro Lys Ala

115

<210> 3

<211> 121

<212> PRT

<213> Artificial sequence

<400> 3

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Leu

20 25 30

Ser Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Gly Phe Asp Arg Glu Asp Gly Glu Thr Ile Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Glu Asp Thr Ser Thr Asp Thr Ala Tyr

65 70 75 80

Met Tyr Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ile Asp Pro Ile Arg Tyr Asn Trp Asn Tyr Gly Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 4

<211> 120

<212> PRT

<213> Artificial sequence

<400> 4

Leu Pro Val Leu Thr Gln Pro Pro Ser Ser Ser Ala Ser Pro Gly Glu

1 5 10 15

Ser Ala Arg Leu Thr Cys Thr Leu Pro Ser Asp Ile Ser Val Ser Ser

20 25 30

Tyr Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Arg Phe

35 40 45

Leu Leu Tyr Tyr Tyr Ser Asp Ser Asp Lys Gly Gln Gly Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Asp Asn Thr Gly Ile

65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys

85 90 95

Met Ile Trp Pro Ser Asn Ala Trp Val Phe Gly Gly Gly Thr Lys Leu

100 105 110

Thr Val Leu Gly Gln Pro Lys Ala

115 120

<210> 5

<211> 125

<212> PRT

<213> Artificial sequence

<400> 5

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Asp Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Leu Tyr Tyr

85 90 95

Cys Ala Ser Leu Pro Tyr Cys Ser Gly Arg Ile Cys Arg Pro Arg Thr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 6

<211> 120

<212> PRT

<213> Artificial sequence

<400> 6

Leu Pro Val Leu Thr Gln Pro Pro Ser Ser Ser Ala Ser Pro Gly Glu

1 5 10 15

Ser Ala Arg Leu Thr Cys Thr Leu Pro Ser Asp Ile Asn Val Gly Ser

20 25 30

Tyr Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Arg Tyr

35 40 45

Leu Leu Tyr Tyr Tyr Ser Asp Ser Asp Lys Gly Gln Gly Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Thr Gly Ile

65 70 75 80

Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys

85 90 95

Met Ile Trp Pro Ser Asn Ala Trp Val Phe Gly Gly Gly Thr Lys Leu

100 105 110

Thr Val Leu Gly Gln Pro Lys Ala

115 120

<210> 7

<211> 118

<212> PRT

<213> Artificial sequence

<400> 7

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Thr Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Ser Asp Tyr Gly Asp Leu Ile Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 8

<211> 110

<212> PRT

<213> Artificial sequence

<400> 8

Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asp Lys Tyr Val

20 25 30

Cys Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr

35 40 45

Gln Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Cys Asp Ser Ser Thr Val Phe

85 90 95

Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala

100 105 110

<210> 9

<211> 130

<212> PRT

<213> Artificial sequence

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Lys Thr Tyr Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr Val Leu Gly Pro Gly Ser Gly Tyr Asp Ser Phe Glu

100 105 110

Asp Phe Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val

115 120 125

Ser Ser

130

<210> 10

<211> 113

<212> PRT

<213> Artificial sequence

<400> 10

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala

<210> 11

<211> 119

<212> PRT

<213> Artificial sequence

<400> 11

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Pro Gly Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Ser Ala Asp Val Tyr Asn Trp Phe Asp Pro Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 12

<211> 114

<212> PRT

<213> Artificial sequence

<400> 12

Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Lys

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His

85 90 95

Leu Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln Pro

100 105 110

Lys Ala

<210> 13

<211> 123

<212> PRT

<213> Artificial sequence

<400> 13

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Arg Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ser Ser Tyr

20 25 30

Trp Ile Ser Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Glu Pro Ser Asp Ser Tyr Thr Asn Tyr Ser Pro Ser Phe

50 55 60

Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Val Arg His Gly Val Asp Tyr Tyr Asp Thr Ser Gly Tyr Tyr Tyr Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 14

<211> 111

<212> PRT

<213> Artificial sequence

<400> 14

Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Trp Ala Ser Gln Gly Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Leu Asn Ser Tyr Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala

100 105 110

<210> 15

<211> 123

<212> PRT

<213> Artificial sequence

<400> 15

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser

50 55 60

Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Gly Ser Val Asp Ser Tyr Gly Leu Asp Ala Phe Asp Ile

100 105 110

Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser

115 120

<210> 16

<211> 111

<212> PRT

<213> Artificial sequence

<400> 16

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

<210> 17

<211> 126

<212> PRT

<213> Artificial sequence

<400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr

20 25 30

Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Tyr Ile Ser Ser Arg Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Ser Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Asp Gly Gly Gly Tyr Gly Ser Trp Trp Asn Gln Asn Trp

100 105 110

Phe Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 18

<211> 113

<212> PRT

<213> Artificial sequence

<400> 18

Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln

1 5 10 15

Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala

20 25 30

Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Thr Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Val Ser

50 55 60

Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Gly Asp Tyr Tyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His

85 90 95

Trp Val Phe Gly Gly Gly Thr Lys Leu Ala Val Leu Gly Gln Pro Lys

100 105 110

Ala

<210> 19

<211> 330

<212> PRT

<213> Artificial sequence

<400> 19

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 20

<211> 101

<212> PRT

<213> Artificial sequence

<400> 20

Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala

1 5 10 15

Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala

20 25 30

Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val

35 40 45

Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser

50 55 60

Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr

65 70 75 80

Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala

85 90 95

Pro Thr Glu Cys Ser

100

<210> 21

<211> 105

<212> PRT

<213> Artificial sequence

<400> 21

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

1 5 10 15

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

20 25 30

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

35 40 45

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

50 55 60

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

65 70 75 80

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

85 90 95

Lys Ser Phe Asn Arg Gly Glu Cys Ser

100 105

<210> 22

<211> 21

<212> PRT

<213> Artificial sequence

<400> 22

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp

20

<210> 23

<211> 316

<212> PRT

<213> Artificial sequence

<400> 23

Glu Asp Pro His Leu Arg Asn Arg Pro Gly Lys Gly His Asn Tyr Ile

1 5 10 15

Asp Gly Met Thr Gln Glu Asp Ala Thr Cys Lys Pro Val Thr Tyr Ala

20 25 30

Gly Ala Cys Ser Ser Phe Asp Val Leu Leu Glu Lys Gly Lys Phe Pro

35 40 45

Leu Phe Gln Ser Tyr Ala His His Arg Thr Leu Leu Glu Ala Val His

50 55 60

Asp Thr Ile Ile Ala Lys Ala Asp Pro Pro Ser Cys Asp Leu Gln Ser

65 70 75 80

Ala His Gly Asn Pro Cys Met Lys Glu Lys Leu Val Met Lys Thr His

85 90 95

Cys Pro Asn Asp Tyr Gln Ser Ala His Tyr Leu Asn Asn Asp Gly Lys

100 105 110

Met Ala Ser Val Lys Cys Pro Pro Lys Tyr Glu Leu Thr Glu Asp Cys

115 120 125

Asn Phe Cys Arg Gln Met Thr Gly Ala Ser Leu Lys Lys Gly Ser Tyr

130 135 140

Pro Leu Gln Asp Leu Phe Cys Gln Ser Ser Glu Asp Asp Gly Ser Lys

145 150 155 160

Leu Lys Thr Lys Met Lys Gly Val Cys Glu Val Gly Val Gln Ala Leu

165 170 175

Lys Lys Cys Asp Gly Gln Leu Ser Thr Ala His Glu Val Val Pro Phe

180 185 190

Ala Val Phe Lys Asn Ser Lys Lys Val Tyr Leu Asp Lys Leu Asp Leu

195 200 205

Lys Thr Glu Glu Asn Leu Leu Pro Asp Ser Phe Val Cys Phe Glu His

210 215 220

Lys Gly Gln Tyr Lys Gly Thr Ile Asp Ser Gly Gln Thr Lys Arg Glu

225 230 235 240

Leu Lys Ser Phe Asp Ile Ser Gln Cys Pro Lys Ile Gly Gly His Gly

245 250 255

Ser Lys Lys Cys Thr Gly Asp Ala Ala Phe Cys Ser Ala Tyr Glu Cys

260 265 270

Thr Ala Gln Tyr Ala Asn Ala Tyr Cys Ser His Ala Asn Gly Ser Gly

275 280 285

Ile Val Gln Ile Gln Val Ser Gly Val Trp Lys Lys Pro Leu Cys Val

290 295 300

Gly Tyr Glu Arg Val Val Val Lys Arg Glu Leu Ser

305 310 315

<210> 24

<211> 429

<212> PRT

<213> Artificial sequence

<400> 24

Cys Ser Glu Leu Ile Gln Ala Ser Ser Arg Ile Thr Thr Cys Ser Thr

1 5 10 15

Glu Gly Val Asn Thr Lys Cys Arg Leu Ser Gly Thr Ala Leu Ile Arg

20 25 30

Ala Gly Ser Val Gly Ala Glu Ala Cys Leu Met Leu Lys Gly Val Lys

35 40 45

Glu Asp Gln Thr Lys Phe Leu Lys Ile Lys Thr Val Ser Ser Glu Leu

50 55 60

Ser Cys Arg Glu Gly Gln Ser Tyr Trp Thr Gly Ser Phe Ser Pro Lys

65 70 75 80

Cys Leu Ser Ser Arg Arg Cys His Leu Val Gly Glu Cys His Val Asn

85 90 95

Arg Cys Leu Ser Trp Arg Asp Asn Glu Thr Ser Ala Glu Phe Ser Phe

100 105 110

Val Gly Glu Ser Thr Thr Met Arg Glu Asn Lys Cys Phe Glu Gln Cys

115 120 125

Gly Gly Trp Gly Cys Gly Cys Phe Asn Val Asn Pro Ser Cys Leu Phe

130 135 140

Val His Thr Tyr Leu Gln Ser Val Arg Lys Glu Ala Leu Arg Val Phe

145 150 155 160

Asn Cys Ile Asp Trp Val His Lys Leu Thr Leu Glu Ile Thr Asp Phe

165 170 175

Asp Gly Ser Val Ser Thr Ile Asp Leu Gly Ala Ser Ser Ser Arg Phe

180 185 190

Thr Asn Trp Gly Ser Val Ser Leu Ser Leu Asp Ala Glu Gly Ile Ser

195 200 205

Gly Ser Asn Ser Phe Ser Phe Ile Glu Ser Pro Gly Lys Gly Tyr Ala

210 215 220

Ile Val Asp Glu Pro Phe Ser Glu Ile Pro Arg Gln Gly Phe Leu Gly

225 230 235 240

Glu Ile Arg Cys Asn Ser Glu Ser Ser Val Leu Ser Ala His Glu Ser

245 250 255

Cys Leu Arg Ala Pro Asn Leu Ile Ser Tyr Lys Pro Met Ile Asp Gln

260 265 270

Leu Glu Cys Thr Thr Asn Leu Ile Asp Pro Phe Val Val Phe Glu Arg

275 280 285

Gly Ser Leu Pro Gln Thr Arg Asn Asp Lys Thr Phe Ala Ala Ser Lys

290 295 300

Gly Asn Arg Gly Val Gln Ala Phe Ser Lys Gly Ser Val Gln Ala Asp

305 310 315 320

Leu Thr Leu Met Phe Asp Asn Phe Glu Val Asp Phe Val Gly Ala Ala

325 330 335

Val Ser Cys Asp Ala Ala Phe Leu Asn Leu Thr Gly Cys Tyr Ser Cys

340 345 350

Asn Ala Gly Ala Arg Val Cys Leu Ser Ile Thr Ser Thr Gly Thr Gly

355 360 365

Thr Leu Ser Ala His Asn Lys Asp Gly Ser Leu His Ile Val Leu Pro

370 375 380

Ser Glu Asn Gly Thr Lys Asp Gln Cys Gln Ile Leu His Phe Thr Val

385 390 395 400

Pro Glu Val Glu Glu Glu Phe Met Tyr Ser Cys Asp Gly Asp Glu Arg

405 410 415

Pro Leu Leu Val Lys Gly Thr Leu Ile Ala Ile Asp Pro

420 425

Claims

1. An antibody or antigen-binding fragment directed against rift valley fever virus glycoprotein Gn, comprising a heavy chain and a light chain; the heavy chain comprises a heavy chain variable region and a heavy chain constant region; the light chain comprises a light chain variable region and a light chain constant region;

the antibody or antigen binding fragment is named as R16, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 9 and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 10.

2. The antibody or antigen-binding fragment of rift valley fever virus glycoprotein Gn of claim 1, wherein the amino acid sequence of the heavy chain constant region of R16 is set forth in SEQ ID No. 19.

3. The antibody or antigen-binding fragment of rift valley fever virus glycoprotein Gn of claim 1 or 2, wherein the amino acid sequence of the light chain constant region of R16 is set forth in SEQ ID No. 20.

4. Nucleic acid encoding the antibody or antigen-binding fragment of rift valley fever virus glycoprotein Gn of any of claims 1-3, wherein the nucleic acid comprises a nucleic acid encoding a heavy chain and a nucleic acid encoding a light chain; the nucleic acid for coding the heavy chain sequentially comprises a CMV promoter sequence, a leader sequence, a sequence for coding a heavy chain variable region and a sequence for coding a heavy chain constant region;

or the nucleic acid encoding the heavy chain comprises a CMV promoter sequence, a connecting sequence, a leader sequence, a sequence encoding the variable region of the heavy chain, a sequence encoding the constant region of the heavy chain and a connecting sequence in sequence.

5. The nucleic acid of claim 4, wherein the nucleic acid encoding the light chain comprises, in order, a CMV promoter sequence, a leader sequence, a sequence encoding a light chain variable region, a sequence encoding a light chain constant region;

or the nucleic acid encoding the light chain comprises a CMV promoter sequence, a connecting sequence, a leader sequence, a sequence encoding a light chain variable region, a sequence encoding a light chain constant region, and a connecting sequence in sequence.

6. A plasmid comprising the nucleic acid of claim 4 or 5.

7. A host cell comprising the plasmid of claim 6.

8. Use of an antibody or antigen-binding fragment thereof against rift valley fever virus glycoprotein Gn according to any one of claims 1 to 3 or a nucleic acid according to claim 4 or 5 or a plasmid according to claim 6 or a host cell according to claim 7 for the preparation of a medicament for the treatment and/or prophylaxis of rift valley fever virus.

9. A pharmaceutical composition comprising a prophylactically effective amount of the antibody or antigen-binding fragment of any one of claims 1-3.

10. A kit comprising the antibody or antigen-binding fragment of rift valley fever virus glycoprotein Gc of any one of claims 1-3, or the nucleic acid of claim 4 or 5, or the plasmid of claim 6, or the host cell of claim 7.