A highly stable PRSSV-GP5 nanobody and its preparation method and application

Abstract

The present invention discloses a highly stable PRSSV-GP5 nanobody and a preparation method and application thereof, and belongs to the technical field of PRSSV vaccine. The PRSSV-GP5 nanobody provided by the present invention has a strong affinity and high stability for the antigen PRSSV-GP5, and the equilibrium dissociation constant Kd is 3.84×10 ‑7 M, the stoichiometric N is 1.13, the enthalpy change ΔH is ‑389.24KJ / mol, and the entropy change ΔS is ‑1183.35J / mol·K; the optimal concentration with the antigen is 5μg / mL, and it still has a certain binding ability with the GP5 protein at a concentration of 0.005μg / mL; the preparation method of the PRSSV‑GP5 nanoantibody provided is simple and easy to operate, and can be used for the preparation of PRSSV‑GP5 nanoantibodies with strong antigen affinity, high stability and low binding concentration.

Description

Technical Field

[0001] The present invention relates to the technical field of PRSSV vaccines, and in particular to a highly stable PRSSV-GP5 nano antibody and a preparation method and application thereof. Background Art

[0002] Nanobodies (Nb) are single-domain antibodies derived from the variable domains of HCAbs of animals such as camels and alpacas. They have the characteristics of small molecular weight, simple structure, good biocompatibility, strong permeability, ability to penetrate the blood-brain barrier, and access to deep hidden targets. They have significant advantages in exploring new drug targets and have broad application prospects in clinical diagnosis and treatment. However, their size characteristics also have disadvantages. Their small size makes them easy to be quickly filtered out of the body by the kidneys, resulting in a short retention time of nanobodies in the body, which seriously affects their therapeutic effects.

[0003] Porcine reproductive and respiratory syndrome virus (PRSSV) has 8 structural proteins, namely GP2, E, GP3, GP4, GP5, M, N, and ORF5a. Studies have shown that Nsp2, GP5, and M proteins all have targets for neutralizing antibodies against PRSSV, and GP5 and M proteins are related to the clearance of PRSSV by the pig immune system. The essence of GP5 is a glycosylated envelope protein. According to the position structure of GP5, it is divided into a signal peptide region, an extracellular region, three transmembrane regions, and an intracellular region at the C-terminus. Among them, there is a deceptive non-neutralizing epitope before the neutralizing epitope in the extracellular region of GP5, which can shield the pig immune system, reduce the body's specific response to the neutralizing epitope, and greatly reduce the body's recognition and clearance efficiency of PRSSV. In 2019, researchers used GP5 as an antigen to prepare a subunit vaccine against PRSSV. It is for this reason that although the immunized animals produce specific antibodies, their protection rate to the body is not ideal. In addition, as a single-stranded positive-sense RNA virus, PRSSV lacks a proofreading mechanism in the replication process compared to DNA viruses, has a faster mutation rate, and has complex infection and immune escape mechanisms. The development of new vaccines for PRSSV faces huge challenges, and there is an urgent need to study new strategies to fight viral infections. Summary of the invention

[0004] The purpose of the present invention is to provide a highly stable PRSSV-GP5 nano antibody and a preparation method and application thereof, so as to provide a nano antibody and a preparation method for developing a PRSSV vaccine with high specific reaction, high protection rate, strong affinity with antigen and good stability.

[0005] To achieve the above objectives, the present invention provides a highly stable PRSSV-GP5 nanobody, wherein the highly stable PRSSV-GP5 nanobody is Nb7, the nucleotide sequence of Nb7 is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0006] A method for preparing the highly stable PRSSV-GP5 nanobody as described above, comprising the following steps:

[0007] S1. Adult male alpacas in good physical condition and not vaccinated six months before the immunization were immunized four times with PRSSV-GP5 antigen by multi-point injection;

[0008] S2, separating peripheral blood lymphocytes of immunized alpacas, extracting RNA and reversely transcribing it into cDNA, using the obtained cDNA as a template for two rounds of nested PCR amplification, and performing electrophoresis on the PCR products and then cutting the gel to recover the VHH fragments;

[0009] S3, using sfiI enzyme to digest the pCANTAB 5e vector and VHH fragment respectively, and then ligating them to obtain the pCANTAB 5e-VHH recombinant vector;

[0010] S4, the pCANTAB 5e-VHH recombinant vector was transferred into ER2738 cells, spread on 2×YTAG solid culture plates, cultured at 37°C overnight, and colonies were collected the next day to obtain the pCANTAB5e-Nb-GP5 primary library;

[0011] S5, using helper phage M13K07 to rescue the constructed pCANTAB5e-Nb-GP5 primary library to obtain a phage display library;

[0012] S6. After four rounds of panning of the phage display library using different concentrations of PRSSV-GP5 protein, the positive clone bacterial solution with the highest absorbance value was selected and sent to the company for sequencing to obtain the PRSSV-GP5-specific nanoantibody sequence - Nb7.

[0013] Preferably, in S1, immunization is performed subcutaneously on the back of alpacas; the injection amount of PRSSV-GP5 antigen is 200 μg / time, and the antigen is diluted with complete Freund's adjuvant for the first immunization and injected after emulsification; the antigen is diluted with incomplete Freund's adjuvant for the second to fourth immunizations; and each immunization is performed at an interval of 2 weeks.

[0014] Preferably, the first round of nested PCR in S2 uses the obtained cDNA as a template, with Call001-F and Call002-R as primers, the sequence of Call001-F is shown in SEQ ID NO.1, and the sequence of Call002-R is shown in SEQ ID NO.2; the second round of nested PCR uses the gel-recovered product of the first round of nested PCR as a template, with VHH-F and VHH-R as primers, the sequence of VHH-F is shown in SEQID NO.3, and the sequence of VHH-R is shown in SEQ ID NO.4.

[0015] Preferably, the system of nested PCR in S2 is 10 μL of 2×Taq Master Mix, 1 μL of 10 μM upstream primer and downstream primer, ≤200 ng of template, and filled to 20 μL with RNase-free water.

[0016] Preferably, the first round of nested PCR amplification program in S2 is 94°C 3min; 94°C 30s, 60°C 30s, 72°C 60s, 30 cycles; 72°C 10min; the second round of nested PCR amplification program is 94°C 3min; 94°C 30s, 60°C 30s, 72°C 30s, 30 cycles; 72°C 10min.

[0017] Preferably, the first round of nested PCR gel recovers a heavy chain antibody fragment of about 700 bp, and the second round of nested PCR gel recovers a VHH fragment of about 400 bp.

[0018] Preferably, the concentrations of PRSSV-GP5 protein in S6 are 20 μg / mL, 10 μg / mL, and 5 μg / mL, respectively.

[0019] An application of the highly stable PRSSV-GP5 nanobody as described above in the preparation of porcine reproductive and respiratory syndrome virus vaccine.

[0020] Therefore, the present invention provides a highly stable PRSSV-GP5 nanobody and its preparation method and application, and its specific technical effects are as follows:

[0021] (1) The PRSSV-GP5 nanobody provided by the present invention has strong affinity and high stability for the antigen PRSSV-GP5, and the equilibrium dissociation constant Kd is 3.84×10 -7 M, stoichiometric N is 1.13, enthalpy change ΔH is -389.24KJ / mol, and entropy change ΔS is -1183.35J / mol·K;

[0022] (2) The optimal concentrations of the PRSSV-GP5 nanoantibody Nb7 and the antigen provided by the present invention are both 5 μg / mL, and it still has a certain binding ability with the GP5 protein at a concentration of 0.005 μg / mL;

[0023] (3) The method for preparing the PRSSV-GP5 nanobody provided by the present invention is simple and easy to operate, and can be used to prepare PRSSV-GP5 nanobody with strong antigen affinity, high stability and low binding concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0025] Figure 1 The electrophoresis results of the nested first-round PCR (A) and the nested second-round PCR (B) in Example 1 of the present invention are shown;

[0026] Figure 2 is the storage capacity determination in Example 1 of the present invention 10 -7 Dilution plate (A), library abundance identification 10 -8 Photograph of the dilution plate (B);

[0027] Figure 3 The helper phage M13K07 in Example 1 of the present invention is diluted to 10 -7 Plate photo (A), rescued phage display library at 10 -10 Photograph of the dilution plate (B);

[0028] Figure 4 These are representative photos of the first round (A), second round (B), third round (C), and fourth round (D) of phage library screening in Example 1 of the present invention;

[0029] Figure 5 is the statistical result of the absorbance values ​​of 96 monoclonal strains randomly selected from the fourth round screening plate in Example 1 of the present invention;

[0030] Figure 6 is the sequencing result of the 20 positive clone bacterial solutions with the highest absorbance values ​​selected in Example 1 of the present invention;

[0031] Figure 7It is the Coomassie brilliant blue staining result of the protein collected under different IPTG concentrations and induction conditions in Example 2 of the present invention; wherein lanes 1-2 are the supernatant of precipitation induced by 37°C and 1 mM IPTG, lanes 3-4 are the supernatant of precipitation induced by 37°C and 0.2 mM IPTG, and lanes 5-8 are the supernatant of precipitation induced by 16°C and 1 mM IPTG;

[0032] Figure 8 These are the protein staining results after different steps in the purification process of the Nb7 antibody in Example 2 of the present invention; wherein, lane 1 is the protein treated with a non-denaturing lysis solution, lane 2 is the protein treated with a flow-through solution, lanes 3-4 are the proteins treated with a cleaning solution, and lanes 5-8 are the proteins treated with an eluent solution;

[0033] Fig. 9 is the Western Blot identification result in Example 2 of the present invention;

[0034] Fig.10 It is the affinity result of Nb7 and PRSSV-GP5 in Example 5 of the present invention, wherein A is the heat loss rate result and B is the isotherm curve;

[0035] Fig.11 The binding force of Nb7 with GP5 antigen after being placed at 37°C for 1-11 days in Example 6 of the present invention; ** indicates that compared with the PBS group P ≤0.01, *** indicates compared with PBS group P ≤0.001;

[0036] Fig.12 The binding force of Nb7 with GP5 antigen after being placed at 56°C for 1-11 days in Example 6 of the present invention; ** indicates that compared with the PBS group P ≤0.01, *** indicates compared with PBS group P ≤0.001. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0038] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0039] The instruments, equipment, reagents and materials used in the examples were obtained through commercial channels, among which the alpacas used in the experiment were provided by the alpaca breeding base of Shanxi Agricultural University; PRSSV-GP5 antigen was purchased from Nanjing Anyan Biotechnology Co., Ltd. with the item number NY-021836P; the phagemid pCANTAB5E vector was donated by the research group of Researcher Zhou Yusen of the Beijing Academy of Military Medical Sciences; ER2738 cells were donated by the research group of Researcher Wang Qihui of the Institute of Microbiology, Chinese Academy of Sciences; and helper phage KM13 was donated by the research group of Researcher Cha Lisha of Anhui Agricultural University.

[0040] Embodiment 1

[0041] Preparation of PRSSV-GP5 specific nanobody, the specific steps are as follows:

[0042] S11. Immunization. Select adult male alpacas (60 kg, 3 years old) in good physical condition and not vaccinated six months before immunization. Use purchased PRSSV-GP5 as antigen and adopt subcutaneous multiple injection for immunization. Inject 400 μL of 1 μg / μL PRSSV-GP5 antigen (200 μg / time) subcutaneously on the back of the alpaca. For the first immunization, use complete Freund's adjuvant to dilute the antigen and emulsify it before injection. For the second to fourth immunizations, use incomplete Freund's adjuvant to dilute the antigen, and each immunization should be 2 weeks apart.

[0043] S12. ELISA plate preparation.

[0044] (1) Dilute the PRSSV-GP5 antigen to 2 μg / mL using the antigen coating solution and mix thoroughly to obtain the diluted antigen solution.

[0045] (2) Slowly add the mixed solution to the ELISA coated plate, add 100 μL of diluted antigen solution to each well, and set up 3 blank wells (without any liquid) and 3 negative control wells (with only coating solution) on each plate.

[0046] (3) Allow the coating to stand overnight at 4°C.

[0047] (4) Remove the ELISA plate that has been coated overnight, discard the coating solution, and spin dry.

[0048] (5) Slowly fill each well with PBS (1×) solution, let it stand for 30 seconds, then discard and shake dry. Repeat this washing process three times. Finally, turn the tube upside down on filter paper to absorb the liquid in the well.

[0049] (6) Slowly add 50 μL of 5% BSA solution (5 g BSA + 100 mL PBS + 0.05% Tween-20) to the reaction wells. This is to block the ELISA plate to reduce nonspecific reactions. No bubbles should be left in the wells during blocking. Incubate at 37°C for 40 min.

[0050] (7) After blocking, slowly fill each well with PBS (1×) solution, let it stand for 30 seconds, then discard and shake dry. Repeat this washing process three times, and finally invert the well onto filter paper to absorb the liquid in the well.

[0051] (8) Seal the ELISA plate and store it at 4°C until required.

[0052] S13. ELISA was used to identify the immune effect.

[0053] Before the first immunization and two weeks after each immunization, procoagulant blood was collected from the immunized alpaca vein, and the serum was separated and marked as: unimmunized serum, one-immunized serum, two-immunized serum, three-immunized serum and four-immunized serum, and stored at -20℃ for subsequent detection. The indirect ELISA method was used to determine the immune effect, which was recorded as unimmunized serum group, one-immunized serum group, two-immunized serum group, three-immunized serum group and four-immunized serum group, and the group without alpaca serum was recorded as the negative group. Each group of serum was diluted 100 times, 1,000 times, 10,000 times and 100,000 times and added to the ELISA plate for detection. Each group was repeated three times and placed in a 37℃ incubator for 1 hour. HRP-labeled llama was used as the secondary antibody and incubated at 37℃ for 1 hour. After adding TMB-hydrogen peroxide urea solution colorimetric solution, it was colorized at 37℃ for 15 minutes in the dark. Finally, the stop solution was added to terminate the reaction, and the absorbance at a wavelength of 450nm was measured by an enzyme marker within 15 minutes.

[0054] The results are shown in Table 1. Compared with the non-immunized alpaca serum group, PRSSV-GP5 antibodies appeared in the alpaca serum after the first immunization, and the absorbance value in the alpaca serum after the second immunization increased significantly, which was significantly different from the non-immunized serum ( P <0.01), the immune effect gradually increased with the increase of immunization times, and the serum after the fourth immunization was diluted 10 -3 After multiple times, it still showed a significant positive reaction, and the immune effect met the needs of subsequent library construction.

[0055] Table 1

[0056] ;

[0057] ** indicates that compared with the non-immunized group P ≤0.01

[0058] S14. Extract RNA from peripheral blood lymphocytes of alpacas and reverse transcribe it into cDNA.

[0059] (1) Two weeks after the fourth immunization, anticoagulated blood was collected from the vein of the immunized alpacas.

[0060] (2) Mix and dilute the freshly collected anticoagulated blood with an equal volume of PBS (1×) by gently pipetting.

[0061] (3) Add the diluted anticoagulated blood into a 50 mL centrifuge tube containing an equal volume of peripheral blood lymphocyte separation medium. Use a Pasteur pipette to carefully and slowly spread the diluted anticoagulated blood on the top of the separation medium. At this time, a clear dividing line will be observed.

[0062] (4) Centrifuge at 800 g for 20 min at room temperature, transfer the upper lymphocyte layer to a new 50 mL centrifuge tube, add 10 mL of 1× PBS to wash, and centrifuge at 6000 g for 10 min at room temperature.

[0063] (5) Discard the supernatant and add the above volume of PBS (1×) to wash the precipitate again. Centrifuge at 6000 g for 5 min.

[0064] (6) Discard the supernatant and resuspend the cells in 1 ml of PBS (1×). Use the Trizol method to extract total RNA from lymphocytes and use a kit to reverse transcribe and synthesize cDNA for later use.

[0065] S15. Amplification and isolation of VHH fragments.

[0066] According to the structural characteristics of the VHH region, the nested PCR method was used to amplify the PRSSV heavy chain variable region. The first round of nested PCR used primers Call001-F (sequence as shown in SEQ ID NO.1) and Call002-R (sequence as shown in SEQ ID NO.2), and the second round of PCR used primers VHH-F (sequence as shown in SEQ ID NO.3) and VHH-R (sequence as shown in SEQ ID NO.4). The second round of PCR products were run on gels for verification and cut out and recovered to obtain the VHH fragment.

[0067] The system of the nested first round PCR is shown in Table 2, the amplification procedure is shown in Table 3, and the results are shown in Table Figure 1 As shown in A, there are two fragments in the nested first round PCR product, namely the traditional antibody fragment at 900 bp and the heavy chain antibody fragment at 700 bp. The heavy chain antibody fragment of about 700 bp was recovered by gel electrophoresis. The system of the nested second round PCR is the same as that in Table 1, except that "cDNA" is replaced by the nested first round PCR gel recovery product, and the primers Call001-F and Call002-R are replaced by primers VHH-F and VHH-R; the amplification program is the same as that in Table 2, except that the extension 4 is 72℃ 30s. The results are shown in Figure 1As shown in B, the amplification product of the nested second-round PCR is a VHH fragment of about 400 bp in size.

[0068] Table 2

[0069] ;

[0070] Table 3

[0071] ;

[0072] SEQ ID NO.1: GTCCTGGCTGCTCTTCTACAAGG

[0073] SEQ ID NO.2: GGTACGTGCTGTTGAACTGTTCC

[0074] SEQ ID NO.3:

[0075] TTTCTATTACTAGGCCCAGCCGGCCAGKTGCAGCTCGTGGAGTCNGGNGG

[0076] SEQ ID NO.4:

[0077] AAACCGTTGGCCATAATGGCCTGGAGCTGGGGTCTTCGCTGTGGT

[0078] S16, VHH phage vector construction.

[0079] The pCANTAB 5e vector and the VHH fragment were digested with sfiI enzyme (NEB), and the digestion system and conditions of the pCANTAB 5e vector are shown in Table 4, and the digestion system and method of the VHH fragment are shown in Table 5. The digested pCANTAB 5e vector and the VHH fragment were connected using the system and method shown in Table 6 to obtain the pCANTAB 5e-VHH recombinant vector.

[0080] Table 4

[0081] ;

[0082] Table 5

[0083] ;

[0084] Table 6

[0085] ;

[0086] S17. Construction of phage display library.

[0087] 1 mL of ER2738 cells was fused with 100 μL of the purified ligation product obtained in step S16 using an electroporation instrument (electroporation parameters: 1800 v, 25 nF, 200 Ω, 5 ms). The fusion product was spread on 20 2×YTAG solid culture plates with a diameter of 90 mm and cultured at 37°C overnight. The next day, the grown plaques were collected with 2×YT liquid culture medium, centrifuged at 4°C and 2000 g for 5 min, the waste liquid was discarded, and the plate was resuspended in 8 mL of 2×YTAG liquid culture medium and labeled as pCANTAB5e-Nb-GP5 primary library.

[0088] Determine the storage capacity: Take 100 μL of the bacterial solution after electroporation and use the gradient dilution method (first add 100 μL of the bacterial solution to 900 μL of SOC medium and mix well, the concentration is 10 -1 , and add 100 μl of the bacterial solution to another tube containing 900 μl of SOC medium, mix well, and the concentration is 10 -2 , diluted successively, the lowest concentration was 10 -8 ), spread the gradient diluted bacterial solution on 2× YTAG plates, culture at 37℃ overnight, count the colonies the next day, and calculate the reservoir capacity. Figure 2 As shown in A, 10 -7 69 monoclonal colonies grew on the dilution plate, and the library capacity was calculated to be 80×69×10 7 = 5.52 × 10 10 .

[0089] Identify the abundance of the library: Take 100 μL of the pCANTAB5e-Nb-GP5 primary library bacterial solution, use the gradient dilution method (same as above), spread the gradient diluted bacterial solution on 2× YTAG plates, culture at 37°C overnight, randomly select 30 monoclonal strains for sequencing, and determine the abundance of the primary library. The results are as follows Figure 2 As shown in B, 10 -8 There are 42 monoclonal colonies on the dilution plate, and the calculated abundance is 10×42×10 8 =4.2×10 10 Pieces / mL.

[0090] Identification of recombination rate: 20 monoclonal strains were randomly selected from the library gradient culture plate, and after shaking the cells in 2× YT medium, the library insertion rate was identified by bacterial liquid PCR, and 30 bacterial liquids were randomly selected for sequencing. The amino acid sequence corresponding to the sequenced nucleotides was analyzed using the Megalign function of DNAStar, and it was found that the amino acid sequence showed obvious constant regions and hypervariable regions, which was consistent with the structural characteristics of nanobodies and was the amino acid sequence of Nb.

[0091] S18, amplification and rescue of helper phage.

[0092] The helper phage M13K07 was diluted in multiples using the double-layer agar method and cultured overnight at 37°C. A single plaque was picked the next day and cultured overnight. The bacterial solution was collected, and the phage was recovered using the PEG / NaCl precipitation method. The titer of the amplified helper phage M13K07 was determined using the gradient dilution method. The results are as follows: Figure 3 As shown in Part A, at a dilution of 10 -7 There were 53 plaques on the plate, and the titer of the prepared M13K07 helper phage was 4.94×10 13 CFU / mL, which can be used for subsequent library panning.

[0093] Take the library bacteria pCANTAB5e-Nb-GP5 and inoculate it into 2×YTAG medium. When it reaches the logarithmic growth phase, add 100 μl of the titer of 1.0×10 10 PFU / mL helper phage, 30 minutes after infection, shake and culture at 37°C, 170rpm overnight. The next day, centrifuge and take the supernatant, add 1 / 5 volume of PEG / NaCl solution and bathe on ice, recover the phage, and measure the titer of the rescued phage display library. The results are as follows: Figure 3 As shown in part B, at 10 -10 There were 32 monoclonal colonies on the dilution plate, and the titer of the rescued phage was 3.2×10 12 CFU / mL, which can be used for subsequent library panning.

[0094] S19, phage library screening.

[0095] Different concentrations of PRSSV-GP5 protein (20μg / mL, 10μg / mL, 5μg / mL) were prepared to coat the immunotubes, and four rounds of specific phage screening were performed using the characteristics of antigen-antibody binding. After each round of screening, the efficiency of phage infection and the titer of amplification were determined using the doubling dilution method. The number of rescued phages was recorded as Input, and the number of eluted phages was recorded as Output. The recovery rate was: Output / Input. The results are shown in Figure 2. Figure 4 As shown in Table 7 , the recovery rate of phages continued to increase after 4 rounds of panning, indicating that GP5-specific Nb was continuously enriched.

[0096] Table 7

[0097] ;

[0098] S20, ELISA identification of positive clones.

[0099] 96 colonies were randomly picked from the 4th round screening plates, inoculated into 2×YTAG medium and cultured at 37°C overnight.

[0100] The next day, 50 μL was taken from the culture medium and inoculated into a new culture medium and shaken to the logarithmic phase. The helper phage was added and the infection was allowed to stand at 37°C for 30 minutes and shaken overnight. The next day, the supernatant was taken as the primary antibody for indirect ELISA detection after centrifugation. 2 μg / mL GP5 was used to coat the ELISA plate, and 2% MPBS was added to block overnight. The next day, the pretreated phage supernatant was added and incubated at 37°C for 2 hours. After washing and patting dry, the secondary antibody was added, washed 3 times, and the color developing solution was added to react in the dark for 15 minutes, and the OD was recorded. 450 The absorbance value of nm is as follows Figure 5 As shown, the absorbance values ​​of the 96 monoclonal strain groups were significantly higher than those of the control group. The results showed that the 96 randomly selected monoclonal strains all contained Nb that could bind to the GP5 antigen.

[0101] Select 20 positive clones with the highest absorbance values ​​and repeat the above steps to prepare phage supernatant. Perform ELISA on the phage supernatant, screen the positive clones and send them to the company for sequencing. Figure 6 As shown, a PRSSV-GP5 specific nano antibody sequence - Nb7 was obtained, the nucleic acid sequence is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0102] SEQ ID NO.5: TTGCAGCTCGTGGAGTCTGGGGGAGACAGTCTGGGGGTTCTTTGAGACTCACGTGCGTTGGCTATGGGTTCGATTTCAACCTCTATGGCATGCACTGGGTTCGCCGGCCTCCAGGAAAGGAACTCGAATACGTCGCTGGCATCACAGCCCGCAGTGACACCACGCACTATGCAGATTCCGTGAAGGGCCGATTCACCGTTTCG AGAGACAACGCCAAGAACACGATGACTCTCGACATGAGCCAACTGAGGCCCGAGGATACGGGCGAGTATTATTGTGCGAGAACCGGGGACCAGACACCCCCGTCCGAGGGCCAGGGAACTCAGGTCACCGTCGCCCCCGGTGAGACTTCGGCCCAGACACCCCCGTCCGAGGGCCAGGGATCTCAGGTCACCGTCCCCCCCGCGCACCAC

[0103] SEQ ID NO.6: LQLVESGGDTVQSGGSLRLTCVGYGFDFNLYGMHWVRRPPGKELEYVAGITARSDTTHYADSVKGRFTVSRDNAKNTMTLDMSQLRPEDTGEYYCARTGDQTPPSEGQGTQVTVAPGETSAQTPPSEGQGSQVTVPPAHH

[0104] Embodiment 2

[0105] The PRSSV-GP5 specific nanobody obtained in Example 1 was expressed, purified and identified, and the specific steps were as follows:

[0106] Construction of prokaryotic expression recombinant plasmids for S21 and Nb.

[0107] The Nb7 gene was connected to the pET-30a(+) prokaryotic expression vector by PCR, and then transformed into DH5α competent cells and spread on LB / Kan plates. 2 to 3 single colonies were picked, the positive single clone colonies were amplified, the plasmids were extracted, and sent to a biological company for sequencing.

[0108] S22 and Nb7 nanobody expression.

[0109] The recombinant plasmid with the correct sequencing results was transformed into BL21 (DE3) competent cells, spread on LB / Kan plates, and cultured in a 37°C constant temperature incubator overnight. A single colony was picked and inoculated into 100 mL LB / Kan medium, and cultured in a constant temperature shaker at 37°C and 220 rpm until the OD of the bacterial solution reached 0. 600 The value was about 0.6. Different concentrations of IPTG were added, and the cells were induced at 37°C, 220rpm for 8h and 16°C, 180rpm for 16h, respectively. An empty vector was set as a control. After the induction, the bacterial solution was collected, centrifuged at 7000rpm for 10min, and the supernatant was discarded. After washing the liquid culture medium with PBS, 20mL of PBS was added to resuspend the cells, and ultrasonically disrupted them in an ice bath (ultrasound for 5s, interval for 10s, 100w), centrifuged at 4°C, 7000rpm for 10min, and the supernatant was collected. Each component was stained with Coomassie Brilliant Blue, and the results were as follows: Figure 7 As shown, Nb7 can be expressed in large amounts in the supernatant under the induction conditions of 16°C and 1 mM IPTG.

[0110] Purification of S23 and Nb7 nanobodies.

[0111] The supernatant after centrifugation was collected and purified using a His tag protein purification kit (P2226, Beyotime). The specific steps are as follows:

[0112] (1) Take 1 mL of nickel column, centrifuge at 4°C, 1000 rpm for 10 seconds, pour out the storage solution, balance with 0.5 mL of non-denaturing lysis buffer, repeat twice, and discard the residual solution. Add 4 mL of lysis buffer supernatant containing the target protein to the gel, shake at 4°C for 60 minutes;

[0113] (2) The mixture is placed in an empty column tube of an affinity chromatography column, the cover at the bottom of the affinity chromatography column tube is opened, and the mixed liquid inside the column is allowed to flow out by the principle of gravity, and the liquid is collected;

[0114] (3) Wash the column 5 to 8 times with 1 mL of non-denaturing washing buffer each time and collect the liquid;

[0115] (4) Elute the target protein 6 to 10 times with 0.5 mL of non-denaturing elution buffer each time. Collect the eluted liquid each time in different centrifuge tubes. This eluate is the purified His-tagged target protein sample. Take each component and stain it with Coomassie Brilliant Blue. The results are as follows: Figure 8 As shown, a relatively single protein band can be observed in the eluate, and the size is consistent with the target protein.

[0116] Identification of S24 and Nb7 nanobodies.

[0117] Pipette 40µL of purified product into a 1.5mL centrifuge tube, add 10µL of 5× protein loading buffer, mix well by pipetting, boil in 100℃ boiling water for 10min, centrifuge at 6000rpm for 5min, and take the supernatant for SDS-PAGE analysis. Mouse anti-His tag monoclonal antibody was used as primary antibody, and HRP-labeled goat anti-mouse IgG was used as secondary antibody for Western Blot identification. The results are as follows: Fig. 9 shown.

[0118] Embodiment 3

[0119] The optimal reaction concentration of PRSSV-GP5 and Nb7 was detected by the chessboard method. The specific steps are as follows:

[0120] (1) Dilute the PRSSV-GP5 antigen in a certain gradient ratio (10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL) and coat the ELISA plate in the order from top to bottom;

[0121] (2) Nb7 was diluted in a certain gradient ratio (10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL) and added from left to right according to ELISA;

[0122] (3) Dilute the secondary antibody at 1:10,000 and then develop the color;

[0123] (4) Detection of OD by microplate reader450nm The results are shown in Table 8. The optimal concentrations of PRSSV-GP5 and Nb7 were both 5 μg / mL.

[0124] Table 8 Optimal antibody concentration detection of Nb7

[0125] ;

[0126] Embodiment 4

[0127] The density gradient dilution method was used to detect the minimum reaction concentration of the purified PRSSV-GP5-specific nanobody and Nb7 in Example 2, and the specific steps were as follows:

[0128] (1) Prepare 100 μL / well of PRSSV-GP5 antigen (5 μg / mL) and coat the ELISA plate at 4°C overnight;

[0129] (2) Nb7 was diluted in a certain gradient (5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.63 μg / mL, 0.315 μg / mL, 0.16 μg / mL, 1.25 μg / mL, 0.08 μg / mL, 0.04 μg / mL, 0.02 μg / mL, 0.01 μg / mL, 0.005 μg / mL, 0.0025 μg / mL) and added from left to right according to ELISA;

[0130] (3) Dilute the secondary antibody at 1:10,000 and then develop the color;

[0131] (4) Detection of OD by microplate reader 450nm The results are shown in Table 9. The minimum reaction concentration of PRSSV-GP5 and Nb7 is 0.0025 μg / mL.

[0132] Table 9 Minimum antibody concentration detection of Nb7 and PRSSV-GP5

[0133] ;

[0134] ** indicates that compared with the PBS group P ≤0.01, *** indicates compared with PBS group P ≤0.001

[0135] Embodiment 5

[0136] The affinity between the purified PRSSV-GP5-specific nanobody and the antigen was investigated by isothermal titration, and the specific steps were as follows:

[0137] (1) Before the experiment, each sample was placed in a vacuum machine for 10 minutes to remove the gas in the sample;

[0138] (2) Use the sampling needle that comes with the instrument to draw up the GP5 antigen and place it in the sample pool as the titrant, and use the titration needle to draw up the antibody as the titrant. The concentration ratio of the titrant to the titrant is 1:10.

[0139] (3) The conditions were set to repeat the injection 20 times at 25°C, with an interval of 200 seconds between each injection. Fig.10 As shown in Figure 2, the equilibrium dissociation constant (Kd) obtained by nanoAnalyzer software was 3.84×10 -7 M, stoichiometry (N) is 1.13, enthalpy (ΔH) is -389.24KJ / mol, and entropy (ΔS) is -1183.35J / mol·K.

[0140] Embodiment 6

[0141] The stability of the purified PRSSV-GP5-specific nanobody in Example 2 was investigated, and the specific steps were as follows:

[0142] Nanobody Nb7 (5 μg / mL and 0.005 μg / mL) was treated using the following different temperature conditions:

[0143] 1) Place at 37℃ for 11 days;

[0144] 2) Place at 56°C for 11 days and detect the binding ability of Nb7 to GP5 antigen by ELISA.

[0145] The results are as follows Fig.11 and Fig.12 As shown, 5 μg / mL and 0.005 μg / mL Nb7 still have binding ability to GP5 antigen after being placed at 37°C and 56°C for 11 days. The above results show that Nb7 has good stability.

[0146] Therefore, the PRSSV-GP5 nanobody provided by the present invention has a strong affinity and high stability for the antigen PRSSV-GP5, and the equilibrium dissociation constant Kd is 3.84×10 -7 M, the stoichiometric N is 1.13, the enthalpy change ΔH is -389.24KJ / mol, and the entropy change ΔS is -1183.35J / mol·K; the optimum concentration with the antigen is 5μg / mL, and it still has a certain binding ability with the GP5 protein at a concentration of 0.0025μg / mL; the preparation method of the PRSSV-GP5 nanoantibody provided is simple and easy to operate, and can be used for the preparation of PRSSV-GP5 nanoantibodies with strong antigen affinity, high stability and low binding concentration.

[0147] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A highly stable PRSSV-GP5 nanobody, characterized in that: The highly stable PRSSV-GP5 nanobody is Nb7, the nucleotide sequence of Nb7 is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.

6.

2. Use of the highly stable PRSSV-GP5 nanobody as claimed in claim 1 in the preparation of porcine reproductive and respiratory syndrome virus vaccine.

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