CN106496326A - A kind of Clec4F nano antibodies and its application - Google Patents

Abstract

The invention provides a Clec4F nanobody and its application, belonging to the technical field of biopharmaceuticals. The amino acid sequence of the Nanobody is shown in SEQ.ID.NO.1, and the present invention also discloses the gene sequence encoding the Nanobody, as shown in SEQ.ID.NO.9, which is encoded by the Nanobody disclosed in the present invention. The sequence can be used to construct expression vectors and host cells expressing nanobodies. The Clec4F nanobody of the present invention can be applied to the research and development of Clec4F molecular detection reagents and as an antagonist for blocking the combination of Clec4F protein molecules and N-acetylgalactosamine molecules, further inhibiting Killing effect of NKT cells.

Description

A kind of Clec4F nanobody and its application

technical field

The invention belongs to the technical field of biopharmaceuticals, and relates to a Clec4F nanobody and the use of the antibody in preparing target-binding Clec4F-positive macrophages and competitively inhibiting the binding of Clec4F and N-acetylgalactosamine (GalNAc) molecules.

Background technique

The full name of the Clec4F protein is C-type lectin domain family 4, member F (Clec4F for short), which is a two-type transmembrane C-type lectin (C type Lectin) containing 550 amino acids, the C-terminal domain of the protein and other C-type Carbohydrate recognition domain homology of lectins. Studies have shown that its protein is concentrated in the expression of liver tissue macrophages, therefore, Clec4F is also often called Kupffer cell (Kupffer cell) receptor.

Clec4F was initially isolated and found in rat liver, and it was thought to be a hepatic fucose-binding lectin at that time. Subsequently, it was found that Clec4F combined with galactose, N-acetylgalactosamine (GalNAc) and glycolipids. Clec4F may play an important role in clearing glycoproteins of galactose and trehalose from blood.

Kupffer cells are the most important group of liver tissue macrophages located in the hepatic sinusoid, and Clec4F can be used as a surface marker to distinguish Kupffer cells from other tissue macrophages and mononuclear phagocytes in the liver. C-type lectins are involved in the presentation of lipid antigens to antigen-presenting cells. NKT cells are a special type of antigen-presenting cells, whose main characteristics are the constancy of T cell receptor gene expression, the restriction of CD1d, and the rapid and high level of cytokine production. NKT cells can both enhance immune response and suppress immune response, thus playing an important role in anti-tumor, anti-infection, suppression of autoimmune diseases and transplantation tolerance. The cells can be activated by a glycolipid molecule called alpha-galactosylceramide (α-GalCer). After being activated, NKT cells can release a large amount of interleukin 4 (IL-4), interferon (IFN-γ) and other cytokines. NKT cells are also considered to be crucial regulatory cells in the immune system. Mouse NKT cells account for 0.5% of total T cell numbers in blood, peripheral lymph nodes, and spleen. While more than 30% of liver T cells have NKT cell phenotype, studies have shown that Kupffer cells in the liver are key NKT cell presenting cells. Kupffer cells can form a stable connection with NKT cells through CD1d, thereby activating NKT cells. At the same time, Clec4F is involved in the presentation of α-GalCer antigen, and α-GalCer connects with CD1d to activate NKT cells. Therefore, Clec4F can be used as a surface marker of Kupffer cells, and it also plays an important role in the presentation of glycolipid antigens. Therefore, the antibody designed to competitively inhibit the combination of Clec4F and N-acetylgalactosamine (GalNAc) molecules will block the activation pathway of NKT cells and alleviate the immune response.

Antibodies and antibody derivatives are the most commonly used methods to study cell surface receptors. Specific antibodies can not only be used in basic experimental research and clinical specimen detection, but also can be used as in vivo tracers of receptors. There are natural heavy chain antibodies lacking light chains in camelids. This antibody only contains a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. The VHH single domain is obtained by phage library screening and monoclonal expression Antibodies are also called nanobodies (Camelid single-domain antibody-fragment, nanobody, Nb). The size of nanobodies is only one tenth of that of ordinary IgG antibodies, and has the advantages of high stability, high affinity and easy labeling. Through more than 20 years of research, nanobodies have been used in multiple research fields as a unique tool for testing and treatment.

No research report on Clec4F nanobody has been seen so far, the main reasons are: the screening of Clec4F nanobody and the degree of immune response of immunized animals to Clec4F recombinant protein, the technical level of constructing phage library and expression vector, and the screening of immune library, etc. related to experimental factors. Screening to obtain nanobodies that not only have high affinity with Clec4F recombinant protein, but also can bind to Clec4F receptor naturally expressed in the liver requires a lot of experimental accumulation. In addition, Clec4F is a newly discovered lectin-responsive protein family molecule, and Clec4F needs to be determined experimentally as a target for constructing nanobodies.

Contents of the invention

The purpose of the present invention is to provide a Clec4F nanobody and its application. The present invention constructs a nanobody for Kupffer cell surface receptor Clec4F, which can be used as a molecular probe for targeting Kupffer cell binding, and can be used to study the role of tissue macrophages in acute and chronic hepatitis and liver cancer, and as a blocker for NKT cell activation agent.

To achieve the above object, the present invention adopts the following technical solutions:

The invention discloses a Clec4F nanobody, which includes a framework region and a complementarity determining region. The nanobody specifically recognizes the Clec4F protein (for example, it can resist mouse Clec4F).

The VHH chain of the Clec4F nanobody includes four framework regions (framework region, FR) FR1～FR4 and three complementarity-determining regions (complementarity-determining region, CDR) CDR1～CDR3; the VHH chain of the Clec4F nanobody The amino acid sequence is shown in SEQ.ID.NO.1.

Wherein, the amino acid sequences of the four framework regions FR1, FR2, FR3 and FR4 are shown in SEQ.ID.NO.2, SEQ.ID.NO.4, SEQ.ID.NO.6 and SEQ.ID.NO.8 respectively The amino acid sequences of the three complementarity determining regions CDR1, CDR2 and CDR3 are respectively shown in SEQ.ID.NO.3, SEQ.ID.NO.5 and SEQ.ID.NO.7.

The present invention discloses the nucleotide sequence encoding the Clec4F nanobody, as shown in SEQ.ID.NO.9. The protein structure encoded by the DNA sequence is selected from: the VHH chain of the Clec4F nanobody or the Clec4F nanobody.

The present invention also provides a prokaryotic expression vector, which contains the above-mentioned nucleotide sequence encoding the Clec4F nanobody. The present invention also provides a prokaryotic host cell containing the above prokaryotic expression vector.

The present invention also discloses the function of the above-mentioned Clec4F nanobody in the detection of inflammation. Specifically, the Clec4F nanobody is used in vitro or in vivo to bind to the Clec4F receptor on the surface of Kupffer cells, so as to monitor the occurrence and development of inflammation. The inflammation includes acute hepatitis, chronic hepatitis, liver cancer and the like.

The present invention also discloses the use of the above-mentioned Clec4F nanobody in the preparation of Clec4F receptor antagonists.

The present invention also relates to the application of the Clec4F nanobody in immunological examination and analysis for non-disease diagnosis and treatment purposes. By using the Clec4F nanobody in the preparation of reagents for adsorbing Clec4F protein, for example, the Clec4F nanobody of the present invention can be made into Clec4F Immunoaffinity column absorbs or enriches Clec4F or enriches receptors or cells that can bind to Clec4F for further research.

The amino acid sequence described in the present invention can be used as a precursor for random, point mutation or humanization transformation to obtain mutants with higher affinity, specificity or stability and lower immunogenicity.

Compared with the prior art, the present invention has the following beneficial technical effects:

In the present invention, through in-depth research on the mechanism of ligand recognition and receptor activation of Clec4F, a member of the C-type lectin family on the surface of Kupffer cells in the liver, the Clec4F nanobody is expressed by phage display technology; High-binding nanobody; the present invention expresses and optimizes Clec4F nanobody protein with high affinity and stable uniformity, which opens up a new field of molecular imaging and NKT cell activity inhibitor targeting Clec4F in hepatitis and liver cancer. It has good research value and application prospect.

Description of drawings

Figure 1 is a DNA electrophoresis image of the Clec4F nanobody phage library.

Fig. 2 is the biopanning result of the Clec4F Nanobody phage display library (check the enrichment rate of Clec4F-specific phage for 4 consecutive biopannings by ELISA).

Figure 3 is a purification and identification diagram of the Clec4F nanobody, Ladder: relative molecular weight standard (kDa).

Fig. 4 is the affinity curve of Clec4F nanobody detected by ELISA.

Figure 5 is the result of measuring the expression of Clec4F in concanavalin A (Concanavalin A, Con A)-induced acute hepatitis in mice by using Nanobodies. The normal mice are shown in the order from left to right and from top to bottom in the figure Liver (Naive), ConA-induced mice produced acute hepatitis 3 hours, 6 hours, 12 hours, 24 hours, 48 hours fusion results of four kinds of staining of mouse liver.

detailed description

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments, which are explanations of the present invention rather than limitations.

According to the expression profile of previous hepatitis and normal liver tissue, the Clec4F molecule was screened out, and the specificity of Clec4F expression to liver macrophages was verified, so as to further confirm that Clec4F was the target for the construction of nanobodies. On this basis, the present invention uses phage display technology to screen the nanobody clone that can specifically bind to the target recombinant protein Clec4F from the heavy chain antibody immunized by alpaca, and use it as a specific probe for Kupffer cells for hepatitis and Molecular probes for liver cancer and inhibitors of NKT cell activity.

The screening route of the nanobody specifically bound to Clec4F adopted in the present invention: 1. Immunize the alpaca with the general Clec4F recombinant protein, separate the lymphocytes in the blood, and use the phage display technology to construct a phage display library; 2. After biological screening 3. After sequencing and biological comparison, use enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) method to screen out high-affinity nanobodies against Clec4F, and use Kupper cell Cells validate the binding capacity of Nanobodies.

1. Construction of Nanobody Library Using Clec4F as Antigen

1) Amplification of VHH gene in alpaca lymphocytes

The Clec4F recombinant protein (Entrez Gene IDs: 51811, Ala65-Fly548 region) obtained by expressing in the eukaryotic expression system of the mouse lymphoma cell line NSO (Shanghai Institute of Biological Sciences, Chinese Academy of Sciences) and purified using the N-terminal His tag (Entrez Gene IDs: 51811, Ala65-Fly548 region) was used as an antigen , the glycosyl group of Clec4F protein enhanced the immunogenicity of the antigen. Alpacas were immunized five times with 100 μg Clec4F recombinant protein, each interval of 3 weeks. During the process, the plasma cells in the immunized animals secreted a large amount of antibodies against Clec4F. After obtaining 50 mL of whole blood, different cell components were stratified by lymph separation fluid to separate lymphocytes. Lymphocyte RNA was extracted by Trizol method, and cDNA was obtained by reverse transcription kit (Invitrogen). Subsequent PCR reactions ensured isolation of the gene encoding the constructed VHH:

The first PCR to obtain the heavy chain antibody gene: use the cDNA of lymphocytes as the PCR template, and add the primers Primer call 01 and Primer call 02 covering the upstream and downstream of IgG involved in the immunoglobulin to amplify different subtypes of IgG, Simultaneously add dNTP, Taq DNA polymerase (Roche) etc. to carry out PCR reaction, 1% agarose gel identifies PCR result, gel recovers the gene band (600-800bps) of IgG2 and IgG3, and measures the DNA concentration of gel recovery.

The second nested PCR obtains the VHH gene and inserts the restriction site at the same time: add Primer A6E and Primer 38 covering the VHH sequence of the IgG2 and IgG3 heavy chain regions as primers, and the upstream and downstream primers are respectively designed with the restriction site Not I and Nco 1, add dNTP, Taq DNA polymerase (Roche) etc. to carry out PCR reaction simultaneously, get a small amount of PCR product and identify the result with 1% agarose gel, obtain the large amount of DNA of VHH used for building a library and purify PCR product with DNA purification kit (Qiagen).

2) Linkage, transfection and library expression of VHH gene and phage vector pHEN4

During the PCR reaction process, primers are added to both ends of the target gene to facilitate the construction of a phage expression pHEN4 plasmid, specifically the pHEN4 phage expression vector (Addgene) and the DNA product obtained by the second PCR, using NotI and NcoI Carry out double digestion with restriction endonucleases, place in a 37°C water bath overnight, identify the digestion results on 1% agarose gel, then add DNA ligase and ligate the DNA and pHEN4 vector overnight, and purify the recombinant plasmid band.

The recombinant plasmids were transformed into TG1 Escherichia coli (Addgene) competent cells cultured overnight by electroporation, and then the transfected host bacteria were cultured in LB liquid medium at 37°C for 1 hour on a shaker, and then separated with LB medium. Perform 1/10, ^1/102 , 1/10 ^{3 -} fold dilution, take 500 μL of the diluted bacterial solution and spread it on the surface of the LB solid culture dish, cultivate TG1 cells overnight in a 37°C incubator, and the next day according to The number of colonies on the plate calculated the library capacity as 2×10 ⁷ .

Randomly select 30 clones, use RP and GIII upstream and downstream primers designed for the restriction site region of the pHEN4 plasmid, and simultaneously add dNTP, Taq DNA polymerase (Roche), etc. to carry out PCR reaction, as shown in Figure 1, after 1% The agarose gel identification confirmed that the insertion rate of the phage display library reached over 74%, which proved that the library was constructed successfully.

2. Use phage display technology to screen phages that bind to Clec4F protein

1) The biological panning link in the phage library is an important step to ensure the acquisition of phages that specifically bind to Clec4F. The present invention has carried out 4 consecutive biological pannings, and the elution step in the panning process removes phages that cannot bind to the Clec4F antigen. Phages capable of binding to the antigen were enriched in each round of panning. The specific operation is to coat (coat) or not coat (uncoat) the Clec4F recombinant protein in two wells of a 96-well plate, and mark it as "+" or "-", and then separate the phage library with the two wells at room temperature. Incubation. After the first round of biopanning was washed several times with PBST buffer (Sigma), the phages that could not bind to Clec4F were removed, and the remaining phages that could bind to Clec4F were dissolved with TEA solution (Sigma) and mixed with helper phage ( Thermo fisher) were infected with TG1 host bacteria at the same time, and after overnight culture, they entered the next round of new screening, and the process was the same as above.

2) ELISA method was used to detect the enrichment rate of biological panning. The Clec4F-coated ELISA plate was blocked with skimmed milk powder, and the phage culture solution of four rounds of biological panning was added respectively, followed by the addition of horseradish peroxidase anti-phage antibody-HRP antibody (Life technology), after incubating at room temperature for 1 hour, the enzyme substrates ABTS and H ₂ O ₂ were added, and the ELISA color development results were measured by spectrophotometer OD405nm. It can be seen from Figure 2 that in each round of screening, the phage expressing VHH The signal of the enzymatic reaction with Clec4F is enhanced, so it can be judged that the Clec4F-specific phage has been enriched in four consecutive rounds of screening.

3. Use ELISA to screen the positive monoclonal binding to Clec4F

Clones were randomly selected from the TG1 bacterial culture dishes of each round of biopanning, cultured in LB medium until the bacterial exponential growth phase was added, and IPTG inducer was added to express the protein overnight. The next day, remove the culture supernatant of each clone, lyse the TG1 cells, add the cell lysate to the Clec4F-coated ELISA plate blocked with skimmed milk powder in advance, and then add anti-HA mouse antibody monoclonal antibody (Covance) , incubated at room temperature for 1 h, and finally added alkaline phosphatase-labeled anti-mouse IgG Ab (Life technology) and alkaline phosphatase substrate, and measured the ELISA color development results by spectrophotometer A405nm. Sequence the positive clones with an absorbance value 3 times higher than the negative control (see Table 1), compare and group the Nanobodies according to the IMGT method according to the sequencing results, and select the expression from the same group of CDR3 in the complementarity-determining region (complementarity-determining region) High quantities of representative Nanobodies were used for follow-up experiments.

Table 1. PE-ELISA statistical results of randomly selected TG1 cell monoclonals in biopanning

Table 1 shows the statistical results of PE-ELISA of randomly selected clones in the second and third rounds of biopanning (Round 2 and Round 3). The numbers in italics in Table 1 show the number of positive clones in the second round of screening, and the positive signal (Specific signal) is high Clones 3 times (>3fold) above the background signal (background) were defined as positive clones.

4. Massive expression and purification of nanobodies

Use Nco I and Eco9II restriction enzymes to carry out double digestion reaction on the pHEN4 plasmid containing the nanobody gene, insert the obtained target gene fragment into the pHEN6 empty plasmid vector (Addgene), and transfect Escherichia coli WK6 (Addgene) competent large intestine bacilli. WK6 was cultured in 1L medium to the growth exponential phase, and the expression of Nanobody was induced by IPTG. After overnight culture, WK6 was collected by centrifugation, and the bacterial lysate containing Nanobody was obtained. Immobilized metal ion affinity chromatography (Immobilized metal ion affinity chromatography, IMAC) affinity chromatography was used for primary purification, and the nanobody with His tag was eluted with 0.5M imidazole, and then the nanobody was purified by size exclusion chromatography (size exclusion chromatography), and then eluted with PBS. The nanobody concentration was determined by A260, and the purity and molecular weight of the nanobody were identified by protein gel electrophoresis and Coomassie brilliant blue staining, and the expression level of the nanobody was calculated (see Figure 3, Table 2).

Table 2. The expression level of Clec4F nanobody in 1 liter of TB medium

In Table 2: 2.22 represents the 22nd in Group 2, 5.10 represents the 10th in Group 5, 6.19 represents the 19th in Group 6, 8.37 represents the 37th in Group 8, 10.16 represents the 16th in Group 10, 12.75 It represents the 75th of the 12th group, and NbBCII10 was used as a negative control.

See Figure 3. After purification by IMAC affinity chromatography and size exclusion chromatography, the purity of Clec4F nanobody and negative control antibody was analyzed by 12% SDS-PAGE gel electrophoresis, and the protein was stained with Coomassie brilliant blue. The results illustrate The size of nanobody is about 15kDa, without other foreign proteins.

5. Determination of the affinity of nanobody and Clec4F protein by ELISA technique

Coat the 96-well plate with Clec4F recombinant protein overnight, block the plate with skimmed milk powder at room temperature for 2 hours, and the VHH chain of the Nanobody (Nb2. ; The amino acid sequence of the VHH chain of the nanobody is shown in SEQ.ID.NO.1) after carrying out 1/2 serial dilution, add the plate and incubate for 1 hour, then add anti-mouse His Ab monoclonal antibody (eBioscience) and alkaline phosphoric acid Enzyme-labeled anti-mouse IgG Ab-AP monoclonal antibody (Roche) was added to the substrate for reaction, and the OD 405nm value was measured with a microwell plate reader.

The Clec4F nanobody that was serially diluted reacted with the Clec4F recombinant protein coated on the ELISA plate, and the A405 value obtained by the experiment was transformed into a logarithmic function with the X=log(X) equation, and then the standard error was taken with the concentration data on the ordinate , and then use the Sigmoidal fitting curve method to draw and fit the ELISA curve, the nanobodies Nb2.22, 5.10, 6.19, 8.37, 10.16 and 12.75 all have binding reactions with the Clec4F recombinant protein, so the enzymatic reaction is positive, and the isotype control The antibody BCII10 did not bind to Clec4F at all, thus proving that the Clec4F nanobody can specifically bind to the Clec4F recombinant protein (see FIG. 4 ).

6. Nanobody was used to determine the expression of Clec4F in mouse concanavalin A (Con A)-induced acute hepatitis.

For example, using nanobody and anti-HA-AF594 (Molecular Probes) in step 5, combined with DAPI, F4/80 (macrophage marker) monoclonal antibody anti-F4/80-AF488 (Invitrogen) or CD31 (endothelial cell marker) Monoclonal antibody anti-CD31-AF647 (Invitrogen) stained mouse liver frozen sections, and the results showed (Fig. 5) that a large number of Kupffer cells were displayed in the normal mouse liver. Kupffer cells were apoptotic after 3 hours, 6 hours and 12 hours, and Kupffer cells reappeared in the liver after 48 hours, which proved that nanobodies can detect the Clec4F marker on the surface of Kupffer cells, so as to observe the damage, repair and organization of the liver in acute hepatitis Regeneration of macrophages.

sequence listing

<110> Xi'an Jiaotong University

<120> A kind of Clec4F nanobody and its application

<160> 9

<210> 1

<211> 121

<212> PRT

<213> Alpaca

<400> 1

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

1 5 10 15

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

16 20 25 30

Thr Tyr Gly Met Thr Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg

31 35 40 45

Glu Phe Val Val Thr Gly Asn Gly Ala Gly Thr Thr Tyr Leu Pro

46 50 55 60

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

61 65 70 75

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

76 80 85 90

Val Tyr Tyr Cys Gly Gly Arg Arg Trp Val Pro Ala Thr Ala Val

91 95 100 105

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

106 110 115 120

Ser

121

<210> 2

<211> 25

<212> PRT

<213> Alpaca

<400> 2

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

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

16 20 25

<210> 3

<211> 8

<212> PRT

<213> Alpaca

<400> 3

Gly Ala Thr Phe Ile Thr Tyr Gly

1 5

<210> 4

<211> 15

<212> PRT

<213> Alpaca

<400> 4

Met Thr Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

1 5 10 15

<210> 5

<211> 8

<212> PRT

<213> Alpaca

<400> 5

Val Thr Gly Asn Gly Ala Gly Thr

1 5

<210> 6

<211> 38

<212> PRT

<213> Alpaca

<400> 6

Thr Tyr Leu Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

1 5 10 15

Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Lys Pro

16 20 25 30

Glu Asp Thr Ala Val Tyr Tyr Cys

31 35

<210> 7

<211> 16

<212> PRT

<213> Alpaca

<400>7

Gly Gly Arg Arg Trp Val Pro Ala Thr Ala Val Asp Gly Val Ala Tyr

1 5 10 15

<210>8

<211>11

<212> PRT

<213> Alpaca

<400>8

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210>9

<211>390

<212>DNA

<213> Synthetic

<400>9

gatgtgcagc tggtggagtc tgggggaggt ttggttcagg ctgggggctc tctgagactc 60

tcctgcgcag cctctggagc cactttcatt acgtatggca tgacctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgtggcagct gtgaccggga atggtgccgg cacaacgtat 180

ctgccctccg tgaagggccg atttaccatc tccagagaca acgccaagaa cacggtgtat 240

ctgcaaatga gcagcctgaa gcctgaggac acggccgttt attackgtgg cgggcggcga 300

tgggtacccg ctactgcagt cgatcaagtc gcgtactggg gccaggggac ccaggtcacc 360

gtctcctcac accaccatca ccatcactaa 390

Claims (10)

1. a kind of Clec4F nano antibodies, it is characterised in that：The aminoacid sequence of the VHH of the nano antibody such as SEQ.ID.NO.1 Shown.

2. a kind of Clec4F nano antibodies according to claim 1, it is characterised in that：The nano antibody is by 4 framework regions FR1, FR2, FR3, FR4, and 3 antigen complementary determining region CDR1, CDR2, CDR3 compositions；4 framework regions FR1, FR2, FR3 With the aminoacid sequence of FR4 respectively as shown in SEQ.ID.NO.2, SEQ.ID.NO.4, SEQ.ID.NO.6 and SEQ.ID.NO.8；3 The aminoacid sequence of individual antigen complementary determining region CDR1, CDR2 and CDR3 respectively such as SEQ.ID.NO.3, SEQ.ID.NO.5 and Shown in SEQ.ID.NO.7.

3. a kind of Clec4F nano antibodies according to claim 2, it is characterised in that：4 framework regions and 3 antigens are mutual Benefit decision area puts in order as FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4, the wherein complementary decision of any one antigen Area is bonded by peptide with adjacent associated frame members area.

4. a kind of Clec4F nano antibodies according to claim 1 or claim 2, it is characterised in that：The nano antibody specificity is known Other Clec4F albumen.

5. a kind of Clec4F nano antibodies according to claim 2, it is characterised in that：The coding nucleotide of the nano antibody Sequence is as shown in SEQ.ID.NO.9 or the coding nucleotide sequence is that nucleotide sequence shown in SEQ.ID.NO.9 is carried out Several nucleotide sequences obtained from nucleotide replacement.

6. a kind of Clec4F nano antibodies expression vector, it is characterised in that：The carrier includes the coding core of Clec4F nano antibodies Nucleotide sequence, the coding nucleotide sequence is as shown in SEQ.ID.NO.9 or the coding nucleotide sequence is right Nucleotide sequence shown in SEQ.ID.NO.9 carries out several nucleotide sequences obtained from nucleotide replacement, and the Clec4F receives Meter Kang Ti is as shown in SEQ.ID.NO.1.

7. a kind of transfection has the host cell of Clec4F nano antibody expression vectors, it is characterised in that：The carrier includes The coding nucleotide sequence of Clec4F nano antibodies, the coding nucleotide sequence is as shown in SEQ.ID.NO.9 or the volume Code nucleotide sequence is to carry out several nucleotides sequences obtained from nucleotide replacement to nucleotide sequence shown in SEQ.ID.NO.9 Row, the Clec4F nano antibodies are as shown in SEQ.ID.NO.1.

8. application of a kind of Clec4F nano antibodies as claimed in claim 1 or 2 in inflammation detectable is prepared.

9. application of a kind of Clec4F nano antibodies as claimed in claim 1 or 2 in Clec4F receptor antagonists are prepared.

10. immunologic test point of a kind of Clec4F nano antibodies as claimed in claim 1 or 2 in non-diseases diagnoses and treatment purpose Analysis or the application in immunologic test analytical reagent is prepared.