A vp2 nanobody and expression vector and application thereof

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a VP2 nanobody, an expression vector thereof and application. The application discloses a VP2 nanobody, wherein the amino acid sequence of the nanobody is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 2. The results of the embodiment show that the molecular weight of the VP2 nanobody is about 15 kDa, which is consistent with the size of the nanobody, and the purified nanobody can be used for preparing a product and / or a medicine for detecting feline distemper.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a VP2 nanobody, an expression vector thereof and application thereof. BACKGROUND

[0002] Single-domain antibody is a specific antibody naturally lacking heavy chain but still having biological activity in Camelidae (llama, camel) and cartilaginous fish. The antigen binding site (VHH) of single-domain antibody has independent antigen recognition ability, and the independently expressed VHH is also called nanobody. Compared with traditional four-chain antibodies, the main features of nanobody are small molecular weight, simple structure, stable physicochemical properties, etc. The excellent properties of nanobody make it have advantages in many aspects: in terms of antibody entering the body, nanobody can pass through some protective barriers in the animal body to enter the disease site to play a role, such as the blood-brain barrier, the blood-testis barrier, etc.; in terms of antigen-antibody binding, it can bind to some hidden epitopes, and is particularly suitable for target sites that are difficult to obtain antibodies, such as GPCRs, ion channels and enzyme active centers, etc.; in terms of reducing production cost, nanobody has simple structure and is easy to express in vitro, and is not easy to produce inclusion bodies, so the production process is simple.

[0003] Feline panleukopenia virus (FPV), also known as feline parvovirus, feline distemper virus, feline infectious enteritis virus, belongs to the parvovirus family and is a single-stranded DNA virus, which encodes two structural proteins VP1 and VP2. The VP2 protein is the main capsid protein and is the main immunoprotective antigen protein of the virus. FPV has a wide range of infection and fast transmission speed, and can infect animals of the cat family, weasel family and raccoon family, etc.; it can cause an acute highly contagious disease, i.e. feline panleukopenia, also known as feline distemper, feline infectious enteritis, feline parvovirus enteritis, feline ataxia, etc. Feline panleukopenia is characterized by high fever, vomiting, dehydration, severe leukopenia and hemorrhagic enteritis, and the infection rate can reach 70%, and the mortality rate can reach 90%. At present, with the increase in the number of pet cats and the increase in the use of cats as experimental animals, the prevention and control of cat diseases need to be highly valued. Therefore, it is urgent to develop a nanobody that can specifically and accurately detect feline panleukopenia virus. SUMMARY

[0004] The purpose of the present application is to provide a VP2 nanobody and its application. The nanobody provided by the present application can not only specifically bind to VP2 protein and be used for ELISA detection of feline distemper, but also has the advantages of small molecular weight, stable physicochemical characteristics, high affinity, simple structure, easy recombinant expression and preparation, and the ability to pass through the blood-brain barrier, etc.

[0005] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0006] The application provides a VP2 nanobody, and an amino acid sequence of the nanobody is shown as SEQ ID NO. 1.

[0007] Preferably, the VP2 nanobody has a molecular weight of 15 kDa.

[0008] Preferably, the VP2 nanobody can specifically bind to a VP2 protein.

[0009] A nucleotide sequence encoding the VP2 nanobody is shown as SEQ ID NO. 2.

[0010] The application also provides a kit for detecting feline panleukopenia virus, and the kit comprises the VP2 nanobody.

[0011] The application also provides a recombinant plasmid comprising the nucleotide sequence.

[0012] The application also provides a yeast expression strain comprising the recombinant plasmid.

[0013] Preferably, the strain is constructed by transforming a yeast strain with a linearized plasmid, and the linearized plasmid is obtained by single enzyme digestion of the recombinant plasmid.

[0014] The application also provides application of the VP2 nanobody in preparing an immunological technique for binding to a VP2 protein.

[0015] The application also provides application of the nanobody or the recombinant plasmid or the expression strain in preparing a product and / or a medicine for detecting feline panleukopenia virus.

[0016] Advantages of the application

[0017] The nanobody screening method provided by the application uses phage display technology for screening, and the phage display technology can display an expressed foreign polypeptide or protein in the form of a fusion protein on the surface of a phage, and then the phage expressing the specific protein is screened through affinity enrichment.

[0018] The VP2 nanobody developed by the application through phage display technology can specifically bind to a VP2 antigen protein.

[0019] The VP2 nanobody can specifically bind to a FPV antigen protein, and can be used for detecting feline panleukopenia.

[0020] The VP2 nanobody has a molecular weight of about 15 kDa, which is consistent with the size of a nanobody, and has the advantages of small molecular weight, stable physicochemical characteristics, high affinity, simple structure, easy recombination expression and preparation, and the ability to pass through a blood-brain barrier. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 For the detection of purified VP2-VHH-His by Western blotting.

[0023] Figure 2 Purified VP2-VHH assay for Coomassie Brilliant Blue detection.

[0024] Figure 3 ELISA using VP2 antigen protein and purified nanobody.

[0025] Figure 4 ELISA of feline panleukopenia serum and purified nanobody. Detailed Implementation

[0026] The present invention provides a feline panleukopenia nanobody, characterized in that the amino acid sequence of the nanobody is VP2-VHH-A.

[0027] QVQLVESGGGLVQPGGALRLSCAASGFNFSEYAMIWVRQAPGKGLE WVSDINSGGDSTYYSDSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYY CTTGGRGSWGQGTHVTVSS(SEQ ID NO.1)

[0028] In the present application, the nucleotide sequence encoding the VP2 Nanobody VP2-VHH-1 : CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGAGCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTCAGTGAATATGCTATGATCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAGATATTAACAGTGGTGGTGATAGCACATACTATTCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTATTGTACCACCGGGGGCCGGGGTTCCTGGGGCCAGGGGACCCACGTCACCGTCTCCTCA (SEQ ID NO. 2)

[0029] In the present application, the optimized nucleotide sequence encoding the VP2 Nanobody VP2-VHH-2: CAGGTCCAGCTGGTCGAGTCAGGAGGAGGTCTTGTTCAGCCAGGAGGTGCTCTTAGATTGTCATGCGCTGCTTCAGGATTCAACTTCTCCGAATACGCTATGATTTGGGTCAGACAAGCTCCAGGAAAGGGTTTGGAGTGGGTCTCCGACATTAACTCCGGTGGAGACTCCACCTATTACTCTGACTCAGTGAAGGGAAGATTCACTATCAGTAGAGATAACGCCAAGAACACTTTGTACCTCCAAATGAACTCCTTGAAGCCAGAGGATACGGCTGTATACTACTGTACTACTGGAGGAAGAGGAAGTTGGGGACAAGGAACACACGTTACCGTTTCATCT (SEQ ID NO. 3)

[0030] The present application optimizes the VP2 nanobody VHH sequence and connects it with the Pichia pastoris vector to construct a Pichia pastoris expression system vector, namely a VP2-VHH-H6 protein expression strain. Specifically, after the VP2-VHH1 strain is selected to extract the plasmid and is connected with the PMD19-T vector, blue-white spot screening is performed, and the sequence company is sent for sequencing to obtain the sequence of VP2-VHH-H6. The sequence is given to the company for sequence optimization, and the optimized sequence is connected with the Pichia pastoris vector, and after being transferred into DH5a, it is sent to the company for sequencing. The plasmid of the strain with successful sequencing is extracted and subjected to single enzyme digestion to obtain a linearized plasmid. The linearized plasmid is transferred into a yeast strain by electroporation to successfully construct a VP2-VHH-H6 protein expression strain, and the VP2 nanobody is induced to express and purified.

[0031] The VP2-VHH-His detection result shows that the molecular weight of the VP2 nanobody is about 15 kDa, which is consistent with the size of the nanobody. The Coomassie brilliant blue detection result shows that the purified VP2 nanobody has single nature. The ELISA result shows that the vp2 nanobody can not only be combined with the vp2 antigen protein, but also be combined with the cat plague positive serum, which further shows that the vp2 nanobody can be used to prepare a product and / or a medicine for detecting cat plague.

[0032] In order to further illustrate the present application, the VP2 nanobody provided by the present application is described in detail below in combination with the drawings and examples, but they cannot be understood as limiting the protection scope of the present application.

[0033] Example 1

[0034] The screening of the VP2 nanobody specifically includes the following steps:

[0035] (1) The cat plague nanobody library is prepared according to the method disclosed in Chinese patent CN201910058785.0, and the first round of panning is performed from the prepared cat plague nanobody library to obtain B16-VP2-VHH1, which is divided and stored at -80℃.

[0036] When panning, 50mM sodium carbonate / sodium bicarbonate buffer is used as the coating buffer, the coating concentration is 20μg / ml, the coating volume is 2ml, and the immune tube is coated with VP2 protein.

[0037] The panning method is as follows:

[0038] 1) 500μl of cat plague nanobody library is inoculated into 100ml of 2×YTAG medium, and cultured at 37℃ with 200rmp shaking for 1 hour to OD 600 0.4;

[0039] 2) Add KM13 helper phage, 100 ml bacterial solution plus 100 μl KM13 helper phage, 37°C, stand for 30 min, then shake for 30 min;

[0040] 3) 4000 x g centrifuge for 10 min, remove the supernatant, resuspend the bacterial pellet with 100 ml 2 x YTAK medium, shake at 200 rpm at 30°C overnight;

[0041] 4) The next morning, centrifuge the bacterial solution at 11000 x g at 4°C for 10 min, transfer the supernatant to a new centrifuge bottle and add 20 ml PEG / NaCl solution, mix well and ice bath for 90 min;

[0042] 5) 11000 x g, 4°C, centrifuge for 30 min, discard the supernatant, then centrifuge again for 2 min, completely aspirate the supernatant;

[0043] 6) Resuspend the pellet with 1.3 ml PBS buffer, then divide it into two 1.5 ml centrifuge tubes, centrifuge at 11600 x g for 10 min;

[0044] 7) Recover the supernatant, name it SR-B16-VP2-VHH1, take 100 μl for titer determination, mix the rest with 1.2 ml MPBS solution, incubate at room temperature for 1 h, get the mixture (VP2-VHH1 treated with MPBS solution), ready for use.

[0045] Coat protein treatment:

[0046] 1) Coat protein, the next day, pour out the liquid in the immunization tube, wash the tube with PBS buffer 3 times.

[0047] 2) Add MPBS to each tube, close at room temperature for 2 h, then wash the tube with PBS buffer 3 times.

[0048] 3) Add 2 ml of the mixture obtained in step 7) of the panning step to the immunization tube, incubate at room temperature for 2 h, then wash the tube with PBST solution 10 times, and then with PBS buffer 10 times.

[0049] 4) Add 2 ml of 100 mM TEA solution to each tube, elute the bound phage by gently shaking at room temperature for 15 min, then add 2 ml of Tris-HCl solution to neutralize.

[0050] 5) Transfer the eluted phage (named SC-B16-VP2-VHH1) to a 50 ml centrifuge tube, and add 16 ml of TG1 bacterial solution with OD 600 0.4, 37°C water bath for 30 min, so that the eluted phage infects the TG1 bacterial solution. (And add 4 ml of OD 600Infect 0.4 of TG1 bacterial broth, final volume 24 ml.

[0051] 6) Take 100 μl of the bacterial broth for titer determination, the rest of the bacterial broth is centrifuged at 4000 g for 10 min.

[0052] 7) Resuspend the bacterial pellet with 1 ml of 2xYT medium, spread the resuspended bacterial broth on 5 2xYT AG solid plates (150 mm plates) and incubate in 30°C incubator overnight.

[0053] 8) The next day, collect the bacterial colonies grown on the plates with 2xYT medium, add 60% glycerol to a final concentration of 15%, which is the primary library bacteria, named B16-VP2-VHH1, aliquot and store at -80°C.

[0054] Determination of the rescued phage titer: Gradient dilution of SR-B16-VP2-VHH1, dilution from 10 -7 ~ 10 -13 ; take 10 μl of the phage to infect 190 μl of TG1 bacterial broth with OD 600 0.4; take 100 μl of the bacterial broth of each dilution to spread on 2xYT AG solid plates, incubate in 30°C incubator overnight; count the colonies on the assay plates, calculate the titer of SR-B16-VP2-VHH1.

[0055] Determination of the eluted phage titer: Gradient dilution of the bacterial broth for titer determination, dilution from 10 -1 ~ 10 -5 ; take 100 μl of the bacterial broth of each dilution to spread on 2xYT AG solid plates, incubate in 30°C incubator overnight; count the colonies on the assay plates, calculate the titer of SC-B16-VP2-VHH1; and then calculate the input / output ratio I / O of the first round of panning.

[0056] On the basis of one round of panning, sequentially perform two to four rounds of panning: VP2 protein coating concentrations are 20 μg / ml, 10 μg / ml, 5 μg / ml, respectively; dilution for determination of the rescued phage titer is 10 -7 ~ 10 -12 , 10 -8 ~ 10 -11 , 10 -8 ~ 10 -11 ; dilution for determination of the eluted phage M13-VP2 titer is 10 -1 ~ 10 -6 , 10 -1 ~ 10 -6 , 10 -8 ~ 10 -11; 200 μl of the eluted phage was neutralized with Tris-HCl solution (1 M, pH 7.4) and used to infect 800 μl of OD 600 TG1 bacteria solution (100 μl was gradient diluted, and the rest was preserved) with OD -3 ~10 -6 Each dilution was plated on 3 2 x YTAG solid plates (150 mm plates) with 100 μl of bacteria solution per plate, and incubated at 30°C overnight. The plates were labeled as plates, and stored in a refrigerator at 4°C for later use.

[0057] Screening of specific nanobodies:

[0058] Preparation of supernatant of monoclonal phage: 96 single colonies were picked from each plate and inoculated into a 96-well deep plate, each well containing 1 ml of 2 x YTAG medium. The plates were labeled as VP2 library strains, and incubated at 30°C with shaking. After 8 h, 20 μl of the bacteria solution was inoculated into 180 μl of 2 x YTAG medium, and incubated at 37°C with shaking. The remaining bacteria solution in the original plate was mixed with 60 μl of 60% glycerol to a final concentration of 15%, and stored at -80°C. After the transfer plate was incubated at 37°C with shaking for 1 h, 50 μl of KM13 (60 μl of KM13 + 12 ml of 2 x YTAG medium) was added to each well to assist phage infection, and incubated at 37°C for 30 min. The plate was then incubated at 37°C with shaking for 40 min. The plate was centrifuged at 1800 x g for 10 min, and the supernatant was discarded. The precipitate was resuspended in 400 μl of 2 x YTAK medium, and incubated at 30°C with shaking overnight. The next day, the plate was centrifuged at the maximum speed of 2020 x g for 20 min. 250 μl of the phage supernatant was transferred to a new deep plate, and 250 μl of blocking solution (3% BSA in PBS buffer) was added to each well. The plate was incubated at room temperature for 1 h, and then used for indirect ELISA detection.

[0059] Identification of specific monoclonal phage: The reactivity of phage supernatant with VP2 protein was detected by indirect ELISA test, and the specific method was as follows: the experimental group, negative control group and BSA control group were designed, VP2 protein was used in the experimental group and negative control group to coat 96-well enzyme-labeled plate, the coating concentration was 2 μg / ml, BSA protein was used in the BSA control group to coat 96-well enzyme-labeled plate, the coating concentration was 2 μg / ml, 100 μl per well, and it was placed at 4°C overnight. The next day, the coating liquid in the hole was discarded, 100 μl of blocking solution was added to each hole at 37°C for 1 hour. Discard the blocking solution in the hole, wash the plate 10 times with PBST. Add 100 μl of secondary antibody (HRP-M13 antibody, dilution 1:6000) to each well, incubate at 37°C for 1 hour. Wash the plate 10 times with PBST. Add 100 μl of color developing substrate to each well, avoid light for 5-15 minutes, then add 50 μl of stop solution to each well to stop the reaction. Place the 96-well enzyme-labeled plate in the plate reader to read the OD 450 absorbance value. The ELISA results were analyzed and the positive strains were determined.

[0060] The positive well was inoculated with glycerol bacteria in 5 ml 2xYTAG medium, and the bacterial solution was sent to the sequencing company for sequencing after 37°C shaking culture. After the sequencing results were returned, the sequencing results were analyzed, the correct strain was selected again, and the above experiment was repeated to verify the positive strain.

[0061] Sequencing and specific monoclonal phage ELISA screening results:

[0062] Sequencing was performed by a sequencing company, and the correct VHH fragment clone strain was selected for indirect ELISA method to detect the reactivity of monoclonal phage supernatant with VP2 protein, VP2 protein was used to coat 96-well enzyme-labeled plate, the coating concentration was 2 μg / ml, 50 μl per well, and it was placed at 4°C overnight. The next day, the coating liquid in the hole was discarded, 100 μl of blocking solution was added to each hole at 37°C for 1 hour. Discard the blocking solution in the hole, wash the plate 10 times with PBST. Select the last column of the 96-well plate as the control group and add 100 μl of PBS, and add 100 μl of blocking solution treated phage supernatant as primary antibody to each of the remaining holes, incubate at 37°C for 1 hour. Wash the plate 10 times with PBST. Add 100 μl of secondary antibody (HRP-M13 antibody, dilution 1:10000) to each well, incubate at 37°C for 1 hour. Wash the plate 10 times with PBST. Add 50 μl of color developing substrate to each well, avoid light for 5 minutes at 37°C, then add 50 μl of stop solution to each well to stop the reaction. Place the 96-well enzyme-labeled plate in the plate reader to read the OD450 Absorbance values. The ELISA results were analyzed and the positive well numbers were determined. Both of these two monoclonal antibodies had different degrees of reactivity with VP2 protein as shown in Table 1.

[0063] Table 1. ELISA screening results of VP2 monoclonal antibodies

[0064]

[0065] The correct sequencing and predicted amino acid sequence is SEQ ID NO. 1 (VP2-VHH-H6).

[0066] Example 2

[0067] Construction of Pichia expression system vector and expression of VP2 nanobody

[0068] One clone with strong positive, VP2-VHH1-H6 (SEQ ID NO. 1), was selected as a specific monoclonal positive strain for subsequent experiments.

[0069] The specific monoclonal was used to construct a subsequent Pichia expression system. The specific method was as follows: after the plasmid of VP2-VHH1 strain was extracted, it was ligated with PMD19-T vector, and after blue-white spot screening, it was sent to a sequencing company for sequencing to obtain the sequence of VP2-VHH-H6. The sequence was given to a company for sequence optimization, and the optimized sequence was ligated with the Pichia vector, and after being transferred into DH5a, it was sent to a company for sequencing. The plasmid of the strain with successful sequencing was extracted and subjected to single enzyme digestion to obtain a linearized plasmid. The linearized plasmid was transferred into a yeast strain by electroporation to successfully construct a VP2-VHH-H6 protein expression strain, and the VP2 nanobody was induced to express and purified.

[0070] Example 3

[0071] Detection of VP2 nanobody

[0072] Purification: the yeast liquid after induction expression was centrifuged to obtain the supernatant, which was purified by His-tag nickel column. A 500 mM imidazole eluent was prepared, and after gradient elution of 3%-100%, it was found that there was a relatively single VP2-VHH-His band in the 40% and 100% imidazole elution gradient.

[0073] VP2-VHH-His detection. The purified nanobodies were used as the protein to be detected for Western blotting identification: 15% separating gel and 5% concentrated gel were prepared, and the supernatant column liquid, combined liquid, and imidazole eluent of different concentrations were added to the lanes, respectively. After electrophoresis, wet transfer was performed, and then blocking was performed at 37°C for 1 h. HRP Anti His-Tag Mouse antibody was prepared at a dilution of 1:20,000, and incubation was performed at 37°C for 1 h. Then, membrane washing was performed with TBST, and exposure was performed. The purified nanobodies were identified. According to the protein band position of the supernatant, and by judging the molecular weight and specific binding, the VP2 nanobodies were identified. The results are shown in Figure 1 The results show that the molecular weight is about 15 kDa, which is consistent with the size of the nanobodies.

[0074] Coomassie brilliant blue detection. The purified nanobodies were used as the protein to be detected for Coomassie brilliant blue identification to determine whether the purified nanobodies were single. 15% separating gel and 5% concentrated gel were prepared, and 40% and 100% imidazole eluent were added to the lanes. After electrophoresis, the gel was stained with Coomassie brilliant blue staining solution. The results are shown in Figure 2 It can be seen that the target band in the original 100% imidazole eluent is relatively single.

[0075] VP2 protein was coated on a 96-well enzyme-labeled plate at a coating concentration of 2 μg / ml, 50 μl per well, and was placed at 4°C overnight. The next day, the coating liquid in the wells was discarded, 100 μl of blocking solution was added to each well, and blocking was performed at 37°C for 1 h. The blocking solution in the wells was discarded, and the plate was washed 10 times with PBST. 50 μl of VP2 nanobodies at a concentration of 1 mg / ml was added to the first well of the first column, and the remaining wells were diluted by 2 times per well. The second and third columns were used as control groups, and 50 μl of PBS was added. Incubation was performed at 37°C for 1 h. The plate was washed 10 times with PBST. 50 μl of secondary antibody (HRP-his Antibody, dilution 1:10,000) was added to each well, and incubation was performed at 37°C for 1 h. The plate was washed 10 times with PBST. 50 μl of color developing substrate was added to each well, and reaction was performed at 37°C for 5 min. Then, 50 μl of stop solution was added to each well to stop the reaction. The 96-well enzyme-labeled plate was placed on a plate reader to read the OD 450 absorption value. The ELISA results are shown in Figure 3 The results show that the vp2 nanobodies can bind to the vp2 antigen protein.

[0076] Cat plague serum coated 96-well enzyme-labeled plate, 50 μl per well, placed in 4℃ overnight. The next day, the coating liquid in the hole was discarded, 100 μl blocking solution was added in each hole, and 37℃ blocking was performed for 1 hour. The blocking solution in the hole was discarded, and the plate was washed 10 times with PBST. 50 μl of VP2 nanobody with a concentration of 1 mg / ml was added to the first hole and the second hole in the first column as a control group. 50 μl of PBS was added to the second column and the third column as a control group, and 37℃ incubation was performed for 1 hour. The plate was washed 10 times with PBST. 50 μl of secondary antibody (HRP-his Antibody, dilution 1:10000) was added in each hole, and 37℃ incubation was performed for 1 hour. The plate was washed 10 times with PBST. 50 μl of color developing substrate was added in each hole, and 37℃ reaction was performed for 5 minutes in the dark. Then, 50 μl of termination liquid was added in each hole to terminate the reaction. The 96-well enzyme-labeled plate was placed on the plate reader to read OD 450 Absorbance value. The ELISA results are shown in Figure 4 The results show that the VP2 nanobody can be combined with cat plague positive serum, and can be used for detection of cat plague virus.

[0077] From the above examples, it can be seen that the VP2 nanobody provided by the application has a molecular weight of about 15 kDa, which meets the size of the nanobody. The purified nanobody can be used for detection of cat plague.

[0078] Although the above examples make a detailed description of the application, it is only a part of the examples of the application, but not all the examples. People can also obtain other examples according to the examples without creativity, and these examples all belong to the protection scope of the application.

Claims

1. A VP2 nanobody, characterized in that, The amino acid sequence of the nanobody is shown as SEQ ID NO.

1.

2. Nanobody according to claim 1, characterized in that, The molecular weight of the VP2 nanobody is 15 kDa.

3. The nanobody of claim 1, wherein The VP2 nanobody can specifically bind to VP2 protein.

4. Nucleotides encoding the VP2 nanobody of claim 1, characterized in that, The nucleotide sequence is shown as SEQ ID NO.

2.

5. A kit for detecting feline panleukopenia virus, characterized by, The kit comprises the VP2 nanobody of claim 1.

6. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleotide sequence of claim 4.

7. A yeast expression strain, characterized in that, The strain comprises the recombinant plasmid of claim 6.

8. The expression strain of claim 7, wherein, The strain is constructed by transforming a yeast strain with a linearized plasmid, which is obtained by single enzyme digestion of the recombinant plasmid of claim 6.

9. Use of the VP2 nanobody of claim 1 in the preparation of an immunoreagent that binds to VP2 protein.

10. Use of the nanobody of claim 1 or the recombinant plasmid of claim 6 or the expression strain of any one of claims 7-8 in the preparation of a product for detecting feline panleukopenia virus.

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