VP2 nano antibody as well as expression vector and application thereof

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a VP2 nano antibody as well as an expression vector and application thereof. The invention discloses a VP2 nano antibody, the amino acid sequence of the nano antibody is as shown in SEQ ID NO. 1, and the nucleotide sequence of the nano antibody is as shown in SEQ ID NO. 2. Results of the embodiment show that the molecular weight of the VP2 nano antibody is about 15kDa and accords with the size of the nano antibody, and the purified nano antibody can be used for preparing products and / or drugs for detecting feline plague.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and specifically relates to a VP2 nano antibody and an expression vector and application thereof. Background Art

[0002] Single-domain antibodies are specific antibodies that are naturally missing heavy chains but still have biological activity in camelids (alpacas, camels) and cartilaginous fish. The antigen binding site (VHH) of single-domain antibodies has independent antigen recognition ability, and independently expressed VHH is also called nanobody. Compared with traditional quadruplex antibodies, the main characteristics of nanobody are: small molecular weight, simple structure, stable physical and chemical properties, etc. The excellent properties of nanobody make it advantageous in many aspects: in terms of antibody entry into the body, nanobody can pass through some protective barriers in the animal body to enter the diseased site to exert its effect, such as blood-brain barrier, blood-testis barrier, etc.; in terms of antigen-antibody binding, it can bind to some hidden antigen epitopes, which is particularly suitable for targets that are difficult to obtain antibodies, such as GPCR, ion channels and enzyme activity centers; in terms of reducing production costs, nanobody has a simple structure and is easy to express in vitro, and it is not easy to produce inclusion bodies in in vitro expression, and the production process is simple.

[0003] Feline panleukopenia virus (FPV), also known as feline parvovirus, feline plague virus, and feline infectious enteritis virus, belongs to the genus Parvovirus of the family Parvoviridae. It is a single-stranded DNA virus that encodes two structural proteins, VP1 and VP2. VP2 protein is the main capsid protein and the main immune protective antigen protein of the virus. FPV has a wide range of infection and a fast transmission speed. It can infect animals such as cats, mustelids, and raccoons. It can cause an acute, highly contagious disease, namely feline panleukopenia, also known as feline plague, feline infectious enteritis, feline parvovirus enteritis, and feline ataxia. The main clinical features of feline panleukopenia are high fever, vomiting, dehydration, severe leukopenia, and hemorrhagic enteritis. The infection rate can reach 70%, and the mortality rate can reach up to 90%. At present, with the increase in the number of pet cats raised and the increase in the number of cats used as experimental animals, great attention should be paid to the prevention and control of cat diseases. Therefore, it is urgent to develop a nano-antibody that can detect feline panleukopenia virus with strong specificity and accuracy. Summary of the invention

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

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

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

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

[0008] Preferably, the VP2 nanobody is capable of specifically binding to the VP2 protein.

[0009] The nucleotide sequence encoding the above-mentioned VP2 nanobody is shown in SEQ ID NO.2.

[0010] The present invention also provides a kit for detecting feline panleukopenia virus, wherein the kit comprises the above-mentioned VP2 nano antibody.

[0011] The present invention also provides a recombinant plasmid, which contains the above nucleotide sequence.

[0012] The present invention also provides a yeast expression strain, which contains the above-mentioned recombinant plasmid.

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

[0014] The present invention also provides the use of the above-mentioned VP2 nanobody in preparing an immune technology that binds to the VP2 protein.

[0015] The present invention also provides the use of the above-mentioned nano antibody or the above-mentioned recombinant plasmid or the above-mentioned expression strain in the preparation of products and / or medicines for detecting feline panleukopenia virus.

[0016] Beneficial effects of the present invention

[0017] The nano-antibody screening method of the present invention utilizes phage display technology for screening. Phage display technology can display the expressed exogenous polypeptide or protein on the surface of phage in the form of fusion protein, and then screen phage expressing specific protein by affinity enrichment method.

[0018] The VP2 nanoantibody developed by the present invention using phage display experimental technology can specifically bind to the VP2 antigen protein.

[0019] The VP2 nanoantibody of the present invention can specifically bind to FPV antigen protein and can be used for the detection of feline panleukopenia.

[0020] The VP2 nanobody described in the present invention has a molecular weight of about 15 kDa, which is consistent with the size of nanobodies, and has the advantages of small molecular weight, stable physicochemical characteristics, high affinity and simple structure, easy recombinant expression and preparation, and the ability to cross the blood-brain barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0022] Figure 1 Western Blotting was used to detect purified VP2-VHH-His.

[0023] Figure 2 Purified VP2-VHH was detected by Coomassie Brilliant Blue.

[0024] Figure 3 It is ELISA of VP2 antigen protein and purified nanobody.

[0025] Figure 4 It is an ELISA of feline panleukopenia serum and purified nanoantibodies. DETAILED DESCRIPTION

[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 invention, the nucleotide sequence encoding the VP2 nanobody VP2-VHH-1: CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAACCTGGGGGAGCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCAACTTCAGTGAATATGCTATGATCTGGGTCCGCCAGGCTCCAGGAAAGGGGCTCGAGTGGGTCTCAGATATTAACAGTGGTGGTGATAGCACATACTATTCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTATTGTACCACCGGGGGCCGGGGTTCCTGGGGCCAGGGGACCCACGTCACCGTCTCCTCA(SEQ ID NO.2)

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

[0030] The present invention optimizes the VP2 nano antibody VHH sequence and connects it to a Pichia vector to construct a Pichia expression system vector, i.e., a VP2-VHH-H6 protein expression strain. Specifically, the VP2-VHH1 strain is selected to extract the plasmid and then connected to the PMD19-T vector, and then sent to a sequencing company for sequencing after blue-white screening to obtain the VP2-VHH-H6 sequence. The sequence is sent to the company for sequence optimization, and the optimized sequence is connected to the Pichia vector, transferred to DH5α and sent to the company for sequencing. The plasmid of the strain that has been successfully sequenced is extracted and single enzyme digestion is performed to obtain a linearized plasmid. The linearized plasmid is transferred into the yeast strain by electroporation, and the VP2-VHH-H6 protein expression strain is successfully constructed, and the VP2 nano antibody is induced to express and purified.

[0031] The VP2-VHH-His test results showed that the molecular weight of the VP2 nanobody was about 15kDa, which was consistent with the size of the nanobody. The Coomassie Brilliant Blue test results showed that the purified VP2 nanobody was monospecific. The ELISA results showed that the vp2 nanobody could not only bind to the vp2 antigen protein, but also to the feline panleukopenia-positive serum, further indicating that the vp2 nanobody could be used to prepare products and / or drugs for the detection of feline panleukopenia.

[0032] In order to further illustrate the present invention, the VP2 nanobody provided by the present invention is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1

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

[0035] (1) A feline panleukopenia nanoantibody library was prepared according to the method disclosed in Chinese patent CN201910058785.0, and the first round of panning was performed from the prepared feline panleukopenia nanoantibody library to obtain B16-VP2-VHH1, which was packaged and frozen at -80°C.

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

[0037] The washing method is as follows:

[0038] 1) Inoculate 500 μl of feline panleukopenia nanoantibody library into 100 ml of 2×YTAG medium and culture at 37°C with shaking at 200 rpm for 1 hour until OD 600 is 0.4;

[0039] 2) Add KM13 helper phage, add 100 μl KM13 helper phage to 100 ml bacterial solution, incubate at 37°C for 30 min, and then shake culture for 30 min;

[0040] 3) Centrifuge at 4000×g for 10 min, remove the culture supernatant, resuspend the bacterial pellet in 100 ml of 2×YTAK medium, and culture at 30°C and 200 rpm overnight;

[0041] 4) The next morning, centrifuge the overnight culture at 11,000 × g, 4°C for 10 min, transfer the supernatant to a new centrifuge bottle, add 20 ml of PEG / NaCl solution, mix well, and place on ice for 90 min;

[0042] 5) Centrifuge at 11,000 × g, 4°C for 30 min, discard the supernatant, and then centrifuge again for 2 min to completely aspirate the supernatant;

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

[0044] 7) Recover the supernatant, named 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 hour, and obtain a mixed solution (VP2-VHH1 treated with MPBS solution) for standby use.

[0045] Coating protein treatment:

[0046] 1) The day after coating, pour out the liquid in the immune tube and wash the tube three times with PBS buffer.

[0047] 2) Fill each tube with MPBS, block for 2 h at room temperature, and then wash the tube three times with PBS buffer.

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

[0049] 4) Add 2 ml of 100 mM TEA solution to each tube, shake gently at room temperature for 15 min to elute the bound phages, and 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 OD 600 TG1 bacterial solution with an OD of 0.4 was placed in a 37°C water bath for 30 minutes to allow the eluted phage to infect the TG1 bacterial solution. (Add 4 ml of OD 600The TG1 bacterial solution with a dilution of 0.4 was used for infection and finally combined to a total volume of 24 ml).

[0051] 6) Take 100 μl of bacterial solution for titer determination, and centrifuge the remaining bacterial solution at 4000 g for 10 min.

[0052] 7) Use 1 ml of 2×YT medium to resuspend the bacterial pellet, spread the resuspended bacterial solution on 5 2×YTAG solid culture plates (150 mm plates), and place them in an incubator at 30°C for overnight culture.

[0053] 8) The next day, 2×YT medium was used to collect the colonies grown on the plate, and 60% glycerol was added to a final concentration of 15%. This was the primary library bacteria, named B16-VP2-VHH1, and was aliquoted and frozen at -80°C.

[0054] Determine the titer of rescued phage: SR-B16-VP2-VHH1 was serially diluted from 10 -7 ~10 -13 ; Take 10 μl of phage from each dilution to infect 190 μl OD 600 The dilution of TG1 was 0.4; 100 μl of the bacterial solution was taken for each dilution and coated on a 2×YTAG solid culture plate, which was then placed in a 30°C incubator for overnight culture; the colonies on the assay plate were counted and the titer of SR-B16-VP2-VHH1 was calculated.

[0055] Determine the titer of eluted phage: dilute the bacterial solution used for titer determination in a gradient manner, with the dilution ranging from 10 -1 ~10 -5 ; Take 100 μl of bacterial solution from each dilution and spread it on 2×YTAG solid culture plates, and culture them in a 30°C incubator overnight; count the colonies on the assay plate and calculate the SC-B16-VP2-VHH1 titer; and then calculate the input-output ratio I / O of the first round of panning.

[0056] Based on the first round of panning, two to four rounds of panning were performed in sequence: the VP2 protein coating concentrations were 20 μg / ml, 10 μg / ml, and 5 μg / ml, respectively; the dilutions for the titer determination of rescued phages were 10 -7 ~10 -12 , 10 -8 ~10 -11 , 10 -8 ~10 -11 ; The titer of eluted phage M13-VP2 was determined by dilution of 10 -1 ~10 -6 , 10 -1 ~10 -6 , 10 -8 ~10 -11After the eluted phage was neutralized with Tris-HCl solution (1 M, pH 7.4), 200 μl of phage was used to infect 800 μl of OD 600 TG1 bacterial solution of 0.4 (100 μl was taken for gradient dilution and the rest was used for bacterial preservation), and then 10 -3 ~10 -6 There are 4 dilutions in total. Each dilution is coated on 3 2×YTAG solid culture plates (150 mm plates), 100 μl of bacterial solution is added to each plate, and culture is carried out at 30°C overnight. The colonies on the culture plates are counted, the titers are calculated, and the culture plates are marked as plates and placed in a 4°C refrigerator for use.

[0057] Screening of specific nanobodies:

[0058] Preparation of monoclonal phage supernatant: 96 monoclonal strains were picked from each plate and inoculated into a 96-well deep-well culture plate. Each well contained 1 ml of 2×YTAG medium. The culture plates were marked as VP2 library strains and cultured at 30°C with shaking. After 8 hours, 20 μl of bacterial solution was taken from each well and inoculated into 180 ul of 2×YTAG medium and cultured at 37°C with shaking. 60 μl of 60% glycerol was added to the remaining bacterial solution of the original plate to a final concentration of 15%, and then frozen at -80°C. After the transfer plate was cultured at 37°C with shaking for 1 hour, 50 μl of KM13 (60 μl KM13 + 12 ml of 2×YTAG medium) helper phage was added to each well, and the infection was allowed to stand at 37°C for 30 minutes, and then cultured at 37°C with shaking for 40 minutes. Centrifuge the deep-well plate at 1800×g for 10 min, discard the supernatant and add 400ul 2×YTAK medium to each well to resuspend the precipitate, and shake and culture at 30°C overnight. The next day, centrifuge at a maximum speed of 2020xg for 20 minutes, aspirate 250μl of phage supernatant from each well and transfer it to a new deep-well plate, and add 250μl of blocking solution (PBS buffer solution containing 3% BSA) to each well and incubate at room temperature for 1 hour before use in indirect ELISA detection.

[0059] Identification of specific monoclonal phages: The reactivity of phage supernatants with VP2 protein was detected by indirect ELISA test. The specific method is as follows: experimental group, negative control group and BSA control group were designed. VP2 protein was used to coat 96-well ELISA plates in the experimental group and negative control group at a coating concentration of 2μg / ml. BSA protein was used to coat 96-well ELISA plates in the BSA control group at a coating concentration of 2ug / ml. 100μl was added to each well and placed at 4℃ overnight. The next day, the coating liquid in the wells was discarded, and 100μl of blocking solution was added to each well to block at 37℃ for 1h. The blocking solution in the wells was discarded, and 100μl of phage supernatants obtained from four rounds of screening treated with blocking solution were added to each well of the experimental group and BSA control group as the primary antibody. The negative control was added with an equal amount of PBS and incubated at 37℃ for 1h. The plate was washed 10 times with PBST washing solution. 100μl of secondary antibody (HRP-M13 Antibody, dilution 1:6000) was added to each well and incubated at 37℃ for 1h. Wash the plate 10 times with PBST. Add 100 μl of color substrate to each well, react for 5-15 min in the dark, and then add 50 μl of stop solution to each well to terminate the reaction. Place the 96-well ELISA plate on a plate reader to read the OD 450 Absorption value. Analyze the ELISA results and determine the positive strains.

[0060] Inoculate the glycerol bacteria corresponding to the positive wells into 5 ml of 2×YTAG medium, culture at 37°C with shaking, and send the bacterial solution to the sequencing company for sequencing. After the sequencing results are returned, analyze the sequencing results, select the strains with correct sequencing, and repeat the above experiment again to verify the positive strains.

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

[0062] Sequencing was performed by a sequencing company, and the indirect ELISA method was used to detect the reactivity of the phage supernatant corresponding to the monoclonal with the VP2 protein by selecting the strain that can correctly express the VHH fragment clone. The VP2 protein was coated on a 96-well ELISA plate at a coating concentration of 2 μg / ml, 50 μl per well, and placed at 4°C overnight. The next day, the coating liquid in the well was discarded, and 100 μl of blocking solution was added to each well and blocked at 37°C for 1 hour. The blocking solution in the well was discarded, and the plate was washed 10 times with PBST washing solution. The last column of the 96-well plate was selected as the control group and 100 μl of PBS was added. 100 μl of phage supernatant treated with blocking solution was added to each of the remaining wells as the primary antibody, and incubated at 37°C for 1 hour. The plate was washed 10 times with PBST washing solution. 100 μl of secondary antibody (HRP-M13 Antibody, dilution 1:10000) was added to each well and incubated at 37°C for 1 hour. The plate was washed 10 times with PBST washing solution. Add 50 μl of color substrate to each well, react at 37°C in the dark for 5 minutes, and then add 50 μl of stop solution to each well to terminate the reaction. Place the 96-well ELISA plate on a plate reader to read the OD450 Absorption value. The ELISA results were analyzed and the positive well numbers were determined. Both monoclonal clones had varying degrees of reactivity with VP2 protein as shown in Table 1.

[0063] Table 1 VP2 monoclonal ELISA screening results

[0064]

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

[0066] Example 2

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

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

[0069] The specific monoclonal clone was subsequently subjected to the construction of a Pichia pastoris expression system. The specific method was as follows: the VP2-VHH1 strain was selected to extract the plasmid and then connected to the PMD19-T vector. After blue-white screening, it was sent to a sequencing company for sequencing to obtain the sequence of VP2-VHH-H6. The sequence was sent to the company for sequence optimization, and the optimized sequence was connected to the Pichia pastoris vector, transferred to DH5α and sent to the company for sequencing. The plasmid of the strain that was successfully sequenced was extracted and single enzyme digestion was performed to obtain a linearized plasmid. The linearized plasmid was transferred into the yeast strain by electroporation, and the VP2-VHH-H6 protein expression strain was successfully constructed, and the VP2 nanobody was induced to express and purified.

[0070] Example 3

[0071] VP2 Nanobody Detection

[0072] Purification: The yeast solution after induced expression was centrifuged to obtain the supernatant, which was purified using a His-tagged nickel column. A 500 mM imidazole elution buffer was prepared, and after a 3%-100% gradient elution, a relatively single VP2-VHH-His target band was found in the 40% and 100% imidazole elution gradients.

[0073] VP2-VHH-His detection. The purified nanoantibody was used as the test protein for Western blotting identification: 15% separation gel and 5% concentration gel were prepared, and the supernatant of the yeast solution, the binding solution and the elution solution of different concentrations of imidazole were added to the lanes respectively. After the electrophoresis, wet transfer was performed, and then blocked at 37°C for 1h. HRPAnti His-TagMouse antibody was prepared at a ratio of 1:20000, incubated at 37°C for 1h, and then washed with TBST and exposed to light to detect and identify the purified nanoantibody. According to the position of the protein band in the supernatant stock solution and the molecular weight and specific binding, it was judged to be VP2 nanoantibody. The results are as follows Figure 1 As shown, the molecular weight was found to be approximately 15 kDa, which is consistent with the size of nanobodies.

[0074] Coomassie Brilliant Blue Detection. The purified nanobody is used as the test protein for Coomassie Brilliant Blue identification to determine whether the purified nanobody is single. Prepare 15% separation gel and 5% concentration gel, add 40% and 100% imidazole eluents to the lanes, and after the electrophoresis, stain the colloid with Coomassie Brilliant Blue staining solution. The results are as follows: Figure 2 As shown in the figure, it can be seen that the target band in the original 100% imidazole eluate is relatively single.

[0075] VP2 protein was coated on a 96-well ELISA plate at a coating concentration of 2 μg / ml, 50 μl per well, and placed at 4°C overnight. The next day, the coating liquid in the wells was discarded, and 100 μl of blocking solution was added to each well and blocked at 37°C for 1 hour. The blocking solution in the wells was discarded, and the plate was washed 10 times with PBST washing solution. 50 μl of VP2 nanoantibody at a concentration of 1 mg / ml was added to the first well of the first column, and the remaining wells were diluted 2 times in each well. 50 μl of PBS was added to the second and third columns as the control group, and incubated at 37°C for 1 hour. Wash the plate 10 times with PBST washing solution. 50 μl of secondary antibody (HRP-hisAntibody, dilution 1:10000) was added to each well and incubated at 37°C for 1 hour. Wash the plate 10 times with PBST washing solution. 50 μl of color substrate was added to each well, and the reaction was incubated at 37°C in the dark for 5 minutes, and then 50 μl of stop solution was added to each well to terminate the reaction. Place the 96-well ELISA plate on a plate reader to read the OD 450 Absorption value. ELISA results are as follows Figure 3 As shown. The results show that vp2 nanobody can bind to vp2 antigen protein.

[0076] The feline panleukopenia serum was coated on a 96-well ELISA plate, 50 μl per well, and placed at 4°C overnight. The next day, the coating liquid in the wells was discarded, and 100 μl of blocking solution was added to each well and blocked at 37°C for 1 hour. The blocking solution in the wells was discarded, and the plate was washed 10 times with PBST solution. 50 μl of VP2 nanoantibody at a concentration of 1 mg / ml was added to the first and second wells of the first column. 50 μl of PBS was added to the second and third columns as the control group, and incubated at 37°C for 1 hour. The plate was washed 10 times with PBST solution. 50 μl of secondary antibody (HRP-hisAntibody, dilution 1:10000) was added to each well and incubated at 37°C for 1 hour. The plate was washed 10 times with PBST solution. 50 μl of chromogenic substrate was added to each well, and the reaction was incubated at 37°C in the dark for 5 minutes, and then 50 μl of stop solution was added to each well to terminate the reaction. The 96-well ELISA plate was placed on a plate reader to read the OD 450 Absorption value. ELISA results are as follows Figure 4 The results showed that VP2 nanoantibody can bind to feline panleukopenia positive serum and can be used for the detection of feline panleukopenia virus.

[0077] From the above examples, it can be seen that the molecular weight of the VP2 nanoantibody provided by the present invention is about 15 kDa, which is consistent with the size of nanoantibodies, and the purified nanoantibodies can be used for the detection of feline panleukopenia.

[0078] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

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

1.

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

3. The nanobody according to claim 1, characterized in that The VP2 nanobody can specifically bind to the VP2 protein.

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

2.

5. A kit for detecting feline panleukopenia virus, characterized in that: The kit comprises the VP2 nanobody according to 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 according to claim 6.

8. The expression strain according to claim 7, characterized in that The strain is constructed by transferring a linearized plasmid into a yeast strain, wherein the linearized plasmid is obtained by single enzyme digestion of the recombinant plasmid according to claim 6.

9. Use of the VP2 nanobody according to claim 1 in preparing an immune technology that binds to the VP2 protein.

10. Use of the nanobody according to claim 1, the recombinant plasmid according to claim 6, or the expression strain according to any one of claims 7 to 8 in the preparation of products and / or medicines for detecting feline panleukopenia virus.

Images