CN108084262A - For the full people source single domain antibody or antigen-binding fragment of zika virus and application - Google Patents

Abstract

The invention belongs to the field of biotechnology, and relates to fully human single-domain antibodies against Zika virus, antigen-binding fragments thereof, and their application in preventing and treating Zika virus-related diseases. The fully human single-domain antibody against Zika virus of the present invention and its antigen-binding fragment are mainly characterized in that the single-domain antibody consists of a complementarity-determining region (complementarity-determining region, which exists in the variable region of the antibody heavy chain gene) CDR) specific gene sequence determined, and obtained effective expression in prokaryotic and eukaryotic cells specifically binding Zika virus envelope glycoprotein E (protein E) DIII antibody, using this antibody CDR region or part or all Genes can be used to modify and produce different forms of genetically engineered antibodies in prokaryotic and eukaryotic cells and any expression system, which can be used to prepare clinical preparations for the prevention or treatment of Zika virus-related diseases.

Description

Fully human single domain antibody or antigen-binding fragment against Zika virus and application

technical field

The invention belongs to the field of biotechnology, and specifically relates to a fully human single-domain antibody against Zika virus, an antigen-binding fragment thereof, and their application in preventing and treating Zika virus-related diseases.

Background technique

The prior art discloses that Zika virus is an arbovirus, which was first found in rhesus monkeys in the Zika jungle in Uganda in 1947, and belongs to the genus Flavivirus (flavivirus) like dengue fever, flavivirus and West Nile virus. division). For a long time, Zika virus has been ubiquitous in tropical areas of African and Asian countries, but it cannot cause widespread infection in the population. Until 2007, Zika virus broke out in Yapu Island, Micronesia Islands in the Western Pacific An epidemic with 185 cases was followed by a larger outbreak in the French Polynesia region of Oceania in 2013-2014, which infected about 32,000 people. According to reports, in 2015, the Zika virus arrived in Brazil, causing nearly 30,000 infections, and then quickly spread to other countries in South America and the Caribbean region of Central and North America, and further spread to 66 countries and regions around the world. Since the first imported case was discovered in my country in February 2016, 12 people have been infected with Zika virus so far. At present, more and more studies have confirmed that Zika virus is a neurotropic virus that can infect human neural stem cells, causing the infected cells to die or grow slowly. This series of evidence shows that Zika virus can delay the development of fetal brain development by infecting and slowing down the development of nerve cells and killing nerve cells, causing neonatal microcephaly. In addition, Zika virus can also attack the human immune system, causing a rare autoimmune disease Guillain-Barre syndrome (Guillain-Barre syndrome, GBS), and another autoimmune disease - acute Disseminated encephalomyelitis. An effective vaccine against Zika virus has not yet been developed, and there is no specific therapeutic drug. Studies have shown that Zika virus has the same structure as other viruses of the flavivirus genus. The envelope protein (E protein) of the flavivirus is mainly responsible for the recognition of the virus and the receptor of the infected host, and mediates the invasion of the virus, so it is a virus of this type. The most important protective antigen. A study found that there is a unique positive charge zone on the surface of the Zika virus E protein through the analysis of the three-dimensional crystal structure of the Zika virus E protein, which may make the Zika virus adsorb differently to the host. As a new type of specific anti-viral infection bioengineering agent, antibodies have always played an important role in anti-infection treatment and emergency passive immunization prevention. Therefore, it is necessary to develop antibodies specific to Zika virus for emergency prevention and treatment It is very necessary and feasible.

The in vitro antiviral neutralization activity of monoclonal antibodies and the in vivo protection of the body against viral attacks have been proved by many experiments, such as anti-hepatitis A virus, Hantaan virus, RSV virus, SARS virus, Henipa virus, Ebola virus and other monoclonal antibodies Antibodies can 100% protect animals from virus attack in vivo, but the molecular weight of the full-length monoclonal antibody is too large (150kDa), which leads to some significant defects in its clinical application. First, the excessive molecular weight causes tissue (such as blood Brain barrier) has poor permeability, which affects the therapeutic effect. Secondly, it cannot reach some targets with steric hindrance (steric hindrance). In addition, the large molecular weight leads to high production costs and high prices, making monoclonal antibody drugs in antiviral therapy. The application in fields such as monoclonal antibodies is still relatively limited, and the single domain antibody (VH), which only includes a heavy chain variable region in the antibody, has a molecular weight of only 12-15kDa, which is about 1/10 of the monoclonal antibody, and is currently the smallest Antibody fragments that retain their antigen-binding properties, therefore, have low production costs and high tissue penetration properties that make it possible to treat neurophagic viruses such as Zika virus.

It can be seen that the development of efficient and specific anti-Zika virus single-domain antibodies is of great significance. These single-domain antibodies can not only be used for rapid detection and diagnosis of Zika virus and Zika virus infection in vitro, but also for human infection with Zika Virus prevention and treatment.

The prior art relevant to the present invention has,

[1] Dick GW, Kitchen SF, Haddow AJ. Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg. 1952; 46(5):509-20.

[2] Cao-Lormeau VM, Musso D. Emerging arboviruses in the Pacific. Lancet. 2014; 384(9954): 1571-2.

[3] Cao-Lormeau VM, Roche C, Teissier A, et al. Zika virus, French polynesia, South pacific, 2013. Emerg Infect Dis. 2014; 20(6): 1085-6.

[4] Tang H, Hammack C, Ogden SC, et al. Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth. Cell Stem Cell. 2016; 18(5):587-90.

[5] Garcez PP, Loiola EC, Madeiro da Costa R, et al. Zika virus impairment growth in human neurospheres and brain organoids. Science. 2016; 352(6287): 816-8.

[6] Mlakar J, Korva M, Tul N, et al. Zika Virus Associated with Microcephaly. N Engl J Med. 2016; 374(10): 951-8.

[7] Cao-Lormeau VM, Blake A, Mons S, et al. Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet. 2016; 387(10027): 1531-9.

[8] Dai L, Song J, Lu X, et al. Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody. CellHost Microbe. 2016; 19(5):696-704.

[9] Dimitrov DS. Therapeutic antibodies, vaccines and antibodyomes. MAbs. 2010; 2(3):347-56.

[10] Ying T, Du L, Ju TW, et al. Exceptionally potent neutralization of Middle East respiratory syndrome coronavirus by human monoclonal antibodies. JVirol. 2014; 88(14): 7796-805.

Contents of the invention

The purpose of the present invention is to provide a fully human single domain antibody against Zika virus or an antigen-binding fragment and its application based on the current state of the art. In particular, it relates to a fully human single-domain antibody that specifically binds to Zika virus envelope glycoprotein E.

The fully human single domain antibody of the present invention includes n301, n302 and n303. The invention also discloses antigen-binding fragments of these antibodies, bispecific antibodies, immune conjugates with effector molecules, and fusion proteins.

The antibodies and compositions disclosed in the present invention can be used, for example, for the prevention and treatment of Zika virus infection in humans.

In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the single domain antibody comprises at least one (such as three) CDR regions of the heavy chain variable region of n301, n302 and n303.

The present invention discloses the amino acid sequence of the heavy chain variable region of the single domain antibody:

SEQ ID NO: 1 is the amino acid sequence of the n301 heavy chain variable region;

SEQ ID NO: 2 is the amino acid sequence of n301 heavy chain variable region CDR1;

SEQ ID NO: 3 is the amino acid sequence of n301 heavy chain variable region CDR2;

SEQ ID NO: 4 is the amino acid sequence of n301 heavy chain variable region CDR3;

SEQ ID NO:5 is the amino acid sequence of the n302 heavy chain variable region;

SEQ ID NO: 6 is the amino acid sequence of n302 heavy chain variable region CDR1;

SEQ ID NO: 7 is the amino acid sequence of n302 heavy chain variable region CDR2;

SEQ ID NO: 8 is the amino acid sequence of n302 heavy chain variable region CDR3.

SEQ ID NO: 9 is the amino acid sequence of the n303 heavy chain variable region;

SEQ ID NO: 10 is the amino acid sequence of n303 heavy chain variable region CDR1;

SEQ ID NO: 11 is the amino acid sequence of n303 heavy chain variable region CDR2;

SEQ ID NO: 12 is the amino acid sequence of n303 heavy chain variable region CDR3.

Nucleic acids encoding these single domain antibodies and antigen-binding fragments are also disclosed in the present invention, and in some embodiments of the present invention, vectors comprising these nucleic acids, and host cells comprising these vectors are disclosed; in further embodiments, Pharmaceutical compositions comprising these single domain antibodies, antigen binding fragments, bispecific antibodies, conjugates, fusion proteins, nucleic acids, plasmids, and vectors are disclosed; in other embodiments, these single domain antibodies, antigen binding fragments , bispecific antibodies, conjugates, fusion proteins, nucleic acids, plasmids, and vectors are used to detect, prevent, or treat Zika virus infection.

In the present invention, the fully human single domain antibody against Zika virus, or an antigen-binding fragment, comprises:

The heavy chain variable region of the antibody comprises amino acid residues 26-33, 51-58, and/or 97-108 of SEQ ID NO: 1; or

The heavy chain variable region of the antibody comprises amino acid residues 26-33, 51-58, and/or 97-103 of SEQ ID NO:5; or

The heavy chain variable region of the antibody contains 26-33, 51-58, and/or 97-103 amino acid residues of SEQ ID NO:9,

Wherein the single domain antibody or antigen-binding fragment specifically binds Zika virus.

In the present invention, the single domain antibody or antigen-binding fragment comprises:

The heavy chain variable region of the antibody comprises amino acid residues 26-33, 51-58, and/or 96-108 of SEQ ID NO: 1; or

The heavy chain variable region of the antibody comprises amino acid residues 26-33, 51-58, and/or 97-103 of SEQ ID NO:5; or

The heavy chain variable region of the antibody comprises amino acid residues 26-33, 51-58, and/or 97-103 of SEQ ID NO:9.

In the present invention, the single domain antibody or antigen-binding fragment, wherein:

The amino acid sequence of the heavy chain variable region is at least 90% identical to SEQ ID NO: 1; or

The amino acid sequence of the heavy chain variable region is at least 90% identical to SEQ ID NO:5; or

The amino acid sequence of the heavy chain variable region is at least 90% identical to SEQ ID NO:9.

In the present invention, the single domain antibody or antigen-binding fragment, wherein the single domain antibody or antigen-binding fragment is fully human.

In the present invention, the single domain antibody or antigen-binding fragment, wherein the single domain antibody is VH, and the antigen-binding fragment is VH.

The present invention also provides a bispecific antibody comprising the above-mentioned single domain antibody or antigen-binding fragment.

In the present invention, the single domain antibody or antigen-binding fragment and the bispecific antibody are coupled to an effector molecule; the immunoconjugate, wherein the effector molecule is a detectable label; the The detectable label is a fluorescent label, a radioactive label, avidin, biotin, or an enzyme; the effector molecule is a toxin or a chemotherapeutic agent, wherein the toxin is Pseudomonas aeruginosa exotoxin or a variant thereof.

The present invention also provides a fusion protein comprising the above-mentioned single domain antibody and a heterologous protein, wherein the heterologous protein is human Fc.

The present invention also provides an isolated nucleic acid molecule, which encodes the above-mentioned single domain antibody or antigen-binding fragment, and the bispecific antibody, and the immunoconjugate, or the fusion protein; A nucleic acid molecule operably linked to a promoter.

The present invention also provides a plasmid containing said nucleic acid molecule, wherein the plasmid is a viral plasmid.

The present invention also provides a host cell capable of transforming the nucleic acid molecule or the plasmid.

The present invention also provides a method for detecting Zika virus in a biological sample, comprising:

Containing the biological sample to be detected, and the single domain antibody or antigen-binding fragment, the bispecific antibody, the immunoconjugate, or the fusion protein described in 3, in an amount sufficient to form an immune complex condition, detecting the presence or absence of an immune complex, wherein the presence of the immune complex indicates the presence of Zika virus in the biological sample;

In the detection method, the samples are derived from humans or from mosquitoes.

The present invention also provides a method for detecting whether a subject is infected with Zika virus, comprising:

Containing a biological sample derived from a subject suspected of being infected with Zika virus, and an effective dose of the single domain antibody or antigen-binding fragment, the bispecific antibody, the immunoconjugate, or the A fusion protein that detects the binding of a single domain antibody, antigen-binding fragment, bispecific antibody, immunoconjugate, or fusion protein to the sample, wherein the single domain antibody, antigen-binding fragment, bispecific antibody, immunoconjugate An increase in the binding of the antibody or fusion protein compared to a negative control indicates that the subject is infected with Zika virus; where the negative control is a reference standard, single domain antibody, antigen-binding fragment, bispecific antibody, immunoconjugate or fusion protein with Combination of samples derived from healthy subjects; in said method, the sample is selected from blood, serum, plasma, sputum, or biopsy samples.

The present invention also provides a pharmaceutical composition, which contains an effective preventive dose of the single domain antibody or antigen-binding fragment, the bispecific antibody, the immunoconjugate, the The fusion protein, the nucleic acid, or the plasmid, and a pharmaceutically acceptable carrier.

The present invention is the first in the world to obtain a fully human single-domain antibody against Zika virus. Using this antibody CDR region or part or whole gene, different forms of genetically engineered antibodies can be engineered and produced in prokaryotic and eukaryotic cells and any expression system, which can prevent or treat diseases related to Zika virus infection clinically.

For ease of understanding, the present invention will be described in detail below through specific drawings and embodiments. It should be pointed out that the specific examples and accompanying drawings are only for illustration. Obviously, those skilled in the art can make various amendments and changes within the scope of the present invention according to the description herein. These amendments and Modifications are also included within the scope of the present invention. In addition, the present invention refers to published documents, which are for the purpose of more clearly describing the present invention, the entire contents of which are incorporated herein by reference as if they had been recited herein in their entirety.

Description of drawings

Figure 1. Detection of enriched specific single domain antibodies (VH) using polyclonal phage ELISA.

Figure 2. Detection of binding ability of specific single domain antibody (VH) to Zika virus envelope glycoprotein E by ELISA.

Figure 3. Detection of binding properties of specific single domain antibody (VH) to Zika virus envelope glycoprotein E by Western Blot and biofilm layer interferometry (BLI).

Figure 4. Detection of the neutralizing ability of VH-hFc fusion protein to live Zika virus.

Detailed ways

Example 1

Production of Zika virus E protein:

The synthetic gene encoding Zika virus E protein DIII was inserted into the expression plasmid, and the expression plasmid was transiently transfected into Expi293 cells to express and purify the EDIII-hFc fusion protein;

Screening of Zika virus-specific single domain antibody (VH):

Using human VH 3-23 subfamily germline antibody as a template, use design and antibody engineering technology to introduce heavy chain CDR regions (CDR1, CDR2, CDR3), construct a super-large fully human single-domain antibody library with an actual measured library capacity of 150 billion (1.5×10 ¹¹ ); use this phage display single-domain antibody library to screen antibodies against biotin-labeled EDIII proteins; the present invention , the biotin-labeled EDIII protein was immobilized on streptavidin-coated magnetic beads, and 10 ¹² phage-displayed antibodies were compared with 5, 4, 2, 1 rounds at room temperature in 1, 2, 3, 4 rounds Micrograms of antigen were incubated for two hours, and 10 ¹² phages were used for each round of screening; polyclonal phage ELISA was used to detect the enrichment of antibodies, and the phages in rounds 1, 2, 3, and 4 were incubated with the coated protein, and anti- The HRP-coupled antibody of the phage was used to detect the binding of the phage to the protein; according to the polyclonal phage ELISA results, a very significant enrichment was obtained after the third and fourth rounds of screening (as shown in Figure 1); therefore, these two rounds of screening were selected to obtain The phages were infected with TG1 cells and clones were randomly selected for monoclonal phage ELISA, and further sequenced to identify enriched single domain antibodies;

Detection of Binding Ability of Specific Single Domain Antibody (VH) to Zika Virus E Protein

According to the sequencing results, 19 clones were selected, and the preparation of their soluble expression products was basically carried out according to the literature, specifically: the plasmids of the 19 clones were transferred into HB2151 competent cells, and a single colony was picked from the overnight-grown ampicillin plate and inoculated into SB Bacterial culture medium, expressed under 30-degree IPTG induction conditions for 12-14 hours; harvest the bacteria and purify the single-domain antibody from them with Ni-TNA, coat the Zika virus E protein on the ELISA plate, and add the single-domain diluted in gradient concentration Antibody incubation, using anti-FLAG tag antibody to detect the binding ability of the single domain antibody to the Zika virus E protein, the ELISA results in Figure 2 show that there are 4 single domain antibody clones that can bind to the E protein very strongly, namely n301 and n302 , n303, and n304; according to the sequencing results, n302 and n304 were identified as the same clone, therefore, n301, n302 and n303 were selected for detailed elaboration;

Binding properties of single domain antibodies n301, n302, n303 to Zika virus E protein

After 4%-12% SDS-PAGE electrophoresis, 5 μg of Zika virus E protein was transferred to PVDF membrane, blocked with 5% skimmed milk powder at room temperature for 2 hours, washed with TBST for 3 times, and then incubated with 100 nM single domain antibody overnight at 4°C. After washing the membrane with TBST, add HRP-labeled anti-FLAG secondary antibody (1:1000) and incubate at room temperature for 1.5h, add ECL chemiluminescence solution and expose on a chemiluminescence imager; the Western Blot results in Figure 3A-3B show n301, n302 and n303 Both can bind to the linear Zika virus E protein;

The heavy chain variable regions of single domain antibodies n301, n302 and n303 were constructed into eukaryotic expression vectors containing hFc regions, the expression plasmids were transiently transfected into Expi293 cells and VH-hFc fusion proteins were purified with Protein G, and the purified ZIKV-EDIII -The hFc protein is immobilized on the activated AR2G optical probe and combined with the antibody in gradient dilution. The whole experimental process is as follows: (1) equilibrate (60s); (2) activate AR2G with EDC/NHS (300s); (3) solidify Antigen (300s); (4) ethanolamine blocking non-specific sites (300s); (5) antibody binding (Kon, 300s); (6) antibody dissociation (Koff, 1500s). For competition experiments, ZIKV-EDIII-hFc was bound to AR2G, and each antibody was bound to the antigen at a concentration of 500nM for 300s, and then the second antibody (500nM) mixed with the first antibody (500nM) was added, Figure 3B and 3C shows that n302 and n303 with homologous sequences have the same KD, which are 6.56nM and 6.16nM respectively, and the dissociation speed is very slow. The experiment was to further understand the binding characteristics of n301, n302, n303 and the E protein of Zika disease. The results showed that in the presence of n303, the binding of n302 to the E protein was significantly reduced, but the binding of n301 to n302 and the E protein was significantly reduced. No obvious effect, the results show that n301 has a unique binding site on EDIII with n302 and n303, and n302 and n303 target the same epitope on the surface of EDIII protein; Table 1 shows. VH-hFc and Zika virus envelope sugar Binding properties of protein E;

Table 1

Detection experiment of VH-hFc fusion protein neutralizing ability to live Zika virus

Infect BHK21 cells with 0.2 μM antibody and Zika live virus respectively, detect cell lysate luciferase after incubation, and compare with the control group without antibody, calculate the neutralization percentage, the results are shown in Figure 4, n301, n302, The overall neutralizing activity of live Zika virus was not high, and the neutralizing activity of n303 was the highest.

SEQUENCE LISTING

<110> Fudan University

<120> Fully human single domain antibody or antigen-binding fragment against Zika virus and its application

<130> 20161123

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<170> PatentIn version 3.3

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Claims (25)

1. a kind of full people source single domain antibody or antigen-binding fragment for zika virus, which is characterized in that include：

The heavy chain variable region of antibody contains SEQ ID NO:1 26-33 positions, 51-58 and/or 97-108 amino acids residues； Or

The heavy chain variable region of antibody contains SEQ ID NO:5 26-33 positions, 51-58 and/or 97-103 amino acids residues； Or

The heavy chain variable region of antibody contains SEQ ID NO:9 26-33 positions, 51-58 and/or 97-103 amino acids residues,

Wherein single domain antibody or antigen-binding fragment specific binding zika virus.

2. by single domain antibody described in claim 1 or antigen-binding fragment, which is characterized in that include：

The heavy chain variable region of antibody contains SEQ ID NO:1 26-33 positions, 51-58 and/or 96-108 amino acids residues； Or

The heavy chain variable region of antibody contains SEQ ID NO:5 26-33 positions, 51-58 and/or 97-103 amino acids residues； Or

The heavy chain variable region of antibody contains SEQ ID NO:9 26-33 positions, 51-58 and/or 97-103 amino acids residues.

3. single domain antibody or antigen-binding fragment as described in claim 1 or claim 2, which is characterized in that

Wherein：

The amino acid sequence of heavy chain variable region and SEQ ID NO:1 has at least 90% phase same sex；Or

The amino acid sequence of heavy chain variable region and SEQ ID NO:5 have at least 90% phase same sex；Or

The amino acid sequence of heavy chain variable region and SEQ ID NO:9 have at least 90% phase same sex.

4. single domain antibody or antigen-binding fragment as any one of claim 1-3, which is characterized in that single domain therein Antibody or antigen-binding fragment are full people sources.

5. single domain antibody or antigen-binding fragment as any one of claim 1-4, which is characterized in that single domain therein Antibody is VH, antigen-binding fragment VH.

6. a kind of bispecific antibody, which is characterized in that contain the single domain antibody or antigen any one of claim 1-5 Binding fragment.

It is 7. double special described in the single domain antibody or antigen-binding fragment or claim 6 as any one of claim 1-5 Heterogenetic antibody, which is characterized in that be coupled with a kind of effector molecule.

8. the immune conjugate as described in claim 7, which is characterized in that effector molecule therein is detectable label.

9. the immune conjugate as described in claim 8, which is characterized in that detectable label therein is fluorescent marker, radiation Property mark, Avidin, biotin or enzyme.

10. the immune conjugate as described in claim 7, which is characterized in that effector molecule therein is toxin or chemotherapeutics.

11. the immune conjugate as described in claim 10, which is characterized in that toxin therein be Pseudomonas Exotoxin or its Variant.

12. a kind of fusion protein, which is characterized in that include the single domain antibody any one of claim 1-5 and heterologous egg In vain.

13. the fusion protein as described in claim 12, which is characterized in that wherein heterologous protein is the Fc of people.

14. separated nucleic acid molecules, which is characterized in that it encodes the single domain antibody or anti-any one of claim 1-5 Former binding fragment, the bispecific antibody described in claim 6, the immune conjugate any one of claim 7-11, Or claim 12 or the fusion protein described in claim 13.

15. the nucleic acid molecules as described in claim 14, which is characterized in that be operably coupled to promoter.

16. a kind of plasmid, which is characterized in that contain the nucleic acid molecules described in claim 14 or claim 15.

17. the plasmid as described in claim 16, which is characterized in that wherein plasmid is a kind of virus particle.

18. a kind of host cell, which is characterized in that the nucleic acid molecules or claim 16-17 described in claim 14 can be converted The plasmid.

A kind of 19. method for detecting zika virus in the biological sample, which is characterized in that it includes：

Contain the single domain antibody or antigen-binding fragment any one of detected biological sample and claim 1-5, power Profit requires the bispecific antibody described in 6, immune conjugate or claim 12 any one of claim 7-11 or Fusion protein described in claim 13, under conditions of being enough to form immune complex, detect the presence of immune complex with No, the wherein presence of immune complex represents that there are zika viruses in biological sample.

20. the method as described in claim 19, which is characterized in that wherein sample source is in the mankind.

21. the method as described in claim 19, which is characterized in that wherein sample source is in mosquito.

A kind of 22. method for detecting subject and whether infecting zika virus, which is characterized in that include：

Appoint in the claim 1-5 of biological sample and effective dose containing the subject from suspected infection zika virus Single domain antibody or antigen-binding fragment described in one, the bispecific antibody described in claim 6 are appointed in claim 7-11 The fusion protein described in immune conjugate or claim 12 or claim 13 described in one detects single domain antibody, antigen The combination of binding fragment, bispecific antibody, immune conjugate or fusion protein and the sample, wherein single domain antibody, antigen The combination of binding fragment, bispecific antibody, immune conjugate or fusion protein increases compared to negative control represents subject's sense Contaminate zika virus.

23. the method as described in claim 22, which is characterized in that wherein negative control is a reference standard, single domain antibody, Antigen-binding fragment, bispecific antibody, immune conjugate or fusion protein and from health volunteer sample combination.

24. as the method described in claim 22 or claim 23, wherein sample is blood, serum, blood plasma, sputum or work Sample product.

25. a kind of Pharmaceutical composition, which is characterized in that any one of the claim 1-5 containing effective preventive dose Single domain antibody or antigen-binding fragment, the bispecific antibody described in claim 6, any one of claim 7-11 Immune conjugate, the fusion protein described in claim 12 or claim 13, the core any one of claim 14-15 Acid or the plasmid any one of claim 16-17 and a kind of pharmaceutical acceptable carrier.