Preparation and application of nano antibody Nb12 targeting fish streptococcus agalactiae SIP protein

Abstract

The invention discloses preparation and application of a nano antibody Nb12 targeting fish streptococcus agalactiae SIP protein, and relates to the technical field of fishery disease prevention and control. The nano antibody which is screened by a phage display technology and takes tilapia mossambica source streptococcus agalactiae SIP protein as a target has a heavy chain variable region VHH; the VHH comprises four skeleton regions FR1, FR2, FR3 and FR4 in an antibody variable region and three complementary determinants CDR1, CDR2 and CDR3. The nano antibody Nb12 provided by the invention has relatively high affinity with streptococcus agalactiae SIP protein, can act on the SIP protein in a targeting manner through the antibody, effectively inhibits proliferation of GBS in a tilapia body, has excellent antibacterial activity, and is applied to disease control of the tilapia.

Description

Technical Field

[0001] The present invention relates to the technical field of fishery disease prevention and control, and in particular to a nano antibody Nb12 targeting fish Streptococcus agalactiae SIP protein and an application thereof. Background Art

[0002] Tilapia (Oreochromis sp.) is one of the most important aquaculture species recommended by the Food and Agriculture Organization of the United Nations (FAO) to the world. In 2023, my country's tilapia farming output reached 1.8168 million tons, making it the world's largest tilapia producer. In recent years, with the expansion of farming scale and the increase in farming density, streptococcal disease, mainly caused by Streptococcus agalactiae (GBS), has broken out in my country's main tilapia farming areas. The harm is so great that it is called the "H1N1" of the tilapia farming industry, causing huge economic losses to the tilapia farming industry. At present, antibiotics are still the main method for preventing and controlling Streptococcus agalactiae disease in the farming environment. However, the long-term use and abuse of antibiotics not only leads to serious drug resistance in Streptococcus agalactiae, but also causes a series of problems such as environmental pollution and food safety.

[0003] In the development of tilapia agalactiae disease, virulence factors of S. agalactiae play an important role in invading and lysing host cells. There are many kinds of virulence factors, which can be divided into two categories: structural products and secretory products. Structural products are generally related to the adhesion and colonization of bacteria, while secretory products are often related to the invasion and damage of body cells and tissues. Among them, surface immunogenic protein (SIP) is a structural product of GBS, located on the surface of bacteria, and participates in the adhesion and colonization of bacteria to host cells and tissues. All serotypes of GBS can express SIP protein, and the SIP gene is highly conserved in GBS, has potential immunogenicity, and is an important candidate protein for Streptococcus agalactiae vaccine antigen.

[0004] In the 1990s, Hamers et al. discovered that there was a kind of heavy-chain antibody (HCAbs) in the serum of camelid animals that naturally lacked the light chain part and only contained the heavy chain part. By cloning the variable region gene of HCAbs in camelid animals, a variable domain of heavy-chain antibodies (VHH) consisting only of the heavy chain variable region can be obtained, also known as nanobodies (Nbs). The single domain of VHH has excellent characteristics such as small molecular weight, strong tissue penetration ability, strong tolerance, high affinity, good water solubility, easy cloning and recombination, high yield and low cost, which has promoted its application in the fields of disease diagnosis and treatment.

[0005] Therefore, it is urgent to utilize the excellent properties of VHH to develop a new green prevention and control method for Streptococcus agalactiae. Summary of the invention

[0006] In view of the above shortcomings, the present invention provides a nano-antibody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish, which can be used to prepare neutralizing antibodies that inhibit Streptococcus agalactiae infection in fish, solving the problem that fish currently lack effective neutralizing antibodies for the prevention and control of Streptococcus agalactiae disease. The specific technical scheme is as follows:

[0007] A nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish, wherein the nanobody Nb12 comprises a heavy chain variable region VHH, wherein the VHH comprises four antibody variable region framework regions FR1, FR2, FR3, FR4 and three complementary determining clusters CDR1, CDR2, CDR3, as follows:

[0008] The amino acid sequence of FR1 is shown in SEQ ID NO. 1, i.e., AVQLVESGGGMVQAGGSLKLSCVAS;

[0009] The amino acid sequence of FR2 is shown in SEQ ID NO. 2, i.e., MGWFRQAQGKEREIIAG;

[0010] The amino acid sequence of FR3 is shown in SEQ ID NO. 3, i.e., YYQDSVKDRFTIFRDNAKNTVFLQMNSLRPEDTGVYYC;

[0011] The amino acid sequence of FR4 is shown in SEQ ID NO. 4, i.e., WGQGTQVTVSS;

[0012] The amino acid sequence of CDR1 is shown in SEQ ID NO. 5, i.e., GRTFSTLG;

[0013] The amino acid sequence of CDR2 is shown in SEQ ID NO. 6, i.e., ISWTGQAT;

[0014] The amino acid sequence of CDR3 is shown in SEQ ID NO.7, i.e., AASRSDYDPLFGS;

[0015] The framework regions FR1-4 and the complementary determining clusters CDR1-3 are arranged alternately in sequence, and the amino acid sequence of the heavy chain variable region VHH is shown in SEQ ID NO: 8, that is:

[0016] AVQLVESGGGMVQAGGSLKLSCVASMGWFRQAQGKEREIIAGYYQDSVKDRFTIFR DNAKNTVFLQMNSLRPEDTGVYYCWGQGTQVTVSSGRTFSTLGISWTGQATAASRSDYD PLFGS.

[0017] The present invention also provides a method for preparing the above-mentioned nanoantibody Nb12 targeting the SIP protein of fish Streptococcus agalactiae, comprising the following steps:

[0018] S1. Use genetic engineering technology to insert the VHH gene into the phage coat protein structural gene. As the phage reproduces, the VHH will be expressed as a fusion protein on the coat protein of the progeny phage.

[0019] S2, then utilizing the affinity interaction between the antigen coated in the fixed medium and the VHH, selectively affinity panning the VHH that specifically binds to the antigen;

[0020] S3. After 2-4 rounds of affinity panning, the phage-positive monoclones obtained are subjected to ELISA assay, and the positive clone strains are expanded and sequenced to obtain the nano-antibody Nb12 of the SIP protein.

[0021] Further, the specific steps of the affinity panning are:

[0022] The coated, purified, and renatured SIP protein was blocked with skimmed milk powder on the immunotube, and the VHH phage display library was added and fully incubated, and then washed 15-25 times with a washing solution to remove the phages that did not specifically bind to the SIP protein; the remaining bound phage particles were eluted with an elution solution, and then 10% AEBSF was added to terminate the elution; finally, the eluted phages were infected with the composite strain, and amplified at 35-40°C overnight after infection, and the amplified phages were precipitated and purified using a PEG / 2.5M NaCl solution, and the precipitate was collected and resuspended with a PBS solution, which was the specifically bound phage particles for the next round of screening.

[0023] Furthermore, the washing solution is a PBS solution containing 0.05% Tween 20 (T solution).

[0024] Furthermore, the eluent is a 0.25 mg / mL Trypsin solution.

[0025] Furthermore, the composite strain is Escherichia coli SS320 strain and M13K07 helper phage.

[0026] The present invention also provides an application of the above-mentioned nanoantibody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish. The nanoantibody Nb12 of the present invention can specifically bind to the SIP protein of Streptococcus agalactiae, and is used to prepare neutralizing antibodies that inhibit Streptococcus agalactiae infection of fish, and is used in the prevention and control of related fishery diseases.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The nano antibody Nb12 prepared by the present invention can target the SIP protein of tilapia Streptococcus agalactiae with high affinity, provides a basis for the development of a new generation of green antibacterial agents, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 The results of SDS-PAGE detection of the expression and purification of the recombinant protein Nb12 in Example 1 of the present invention; Lane 1 is the empty vector of E. coli BL21; Lane 2 is the whole E. coli after induced expression; Lane 3 is the supernatant after bacterial liquid crushing and centrifugation after induced expression; Lane 4 is the precipitate after bacterial liquid crushing and centrifugation; Lane P is the purified Nb12;

[0031] Figure 2 This is the affinity test result of the nanobody Nb12 and the target protein SIP in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Unless otherwise defined, all professional terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention. Unless otherwise specifically stated, various raw materials, reagents, instruments and equipment used in the present invention, etc. can be purchased from the market or can be prepared by existing methods.

[0033] Example 1

[0034] 1. VHH selection targeting GBS-SIP

[0035] (1) First round of screening. 50 ug of purified and renatured SIP protein (prepared and stored in the inventor's laboratory) was coated on the immunotube overnight at 4°C. After blocking with 2 mL of 5% (w / v) skim milk powder, 100 ul of naive phage display library (Kangti Life, Shenzhen, China) was added and incubated at 37°C for 1 hour. Unbound phages were then washed 20 times with 2 mL of PBS solution T (1x PBS solution containing 0.05% Tween 20). The remaining bound phage particles were eluted with 1 mL of 0.25 mg / mL Trypsin solution for 30 minutes, and then 10 μL of 10% AEBSF was added to terminate the elution. Finally, the eluted phage was added to infect the Escherichia coli SS320 strain, and the M13K07 helper phage was added for infection and amplified overnight at 37°C. The amplified phage was precipitated and purified using PEG / 2.5M NaCl solution, and the precipitate was collected and resuspended with PBS solution to obtain the collected phage particles. The phage titer was determined and used for the next round of screening.

[0036] (2) Second and third rounds of screening. The remaining steps remained the same except that the phages added to the immune tubes were all purified and amplified after the previous round of screening. The phage titers were measured after each round of screening.

[0037] (3) Monoclonal ELISA assay. Add the purified SIP protein to a 96-well ELISA plate (4 ng / ul, coating solution is PBS solution, pH 8.0, 100ul per well) and coat overnight at 4°C. Wash the ELISA plate three times with PBS solution T, add blocking solution (2.5% skim milk powder) and block at room temperature for 1 hour. Add the phage library and blocking solution at a ratio of 1:9 (the phage library obtained after the third round of panning amplification: blocking solution) and incubate at 37°C for 2 hours. Then add 100ul Anti-VHH Antibody (HRP) diluted 1:5000 in blocking solution to each well and incubate at 37°C for 1 hour. Add 100ul TMB two-component colorimetric solution and color at 37°C in the dark for 15 minutes. Finally, add 50ul ELISA stop solution to stop color development and measure the OD450 value with an ELISA reader. The positive clone strains were expanded and sent to Shanghai Bioengineering for sequencing analysis. By comparing the sequenced sequences, the anti-SIP nanoantibody Nb12 was obtained.

[0038] 2. Construction and identification of pET28a-Nb12 recombinant expression vector

[0039] Using the positive clone strain Nb12 obtained by the above preparation method as a template, amplification primers were designed according to the VHH gene sequence as shown in Table 1:

[0040] Table 1 VHH amplification primers

[0041]

[0042] The PCR amplification reaction system is shown in Table 2:

[0043] Table 2 PCR amplification reaction system

[0044]

[0045]

[0046] The PCR reaction program is shown in Table 3:

[0047] Table 3 PCR reaction program

[0048]

[0049] The PCR product was detected by 1% agarose gel electrophoresis, ligated to the pET28a plasmid through the restriction endonuclease sites EcoRI and Hind III, transferred into Escherichia coli competent cells BL21 (DE3) by thermal transformation, and plated on a kan + Cultured overnight at 37°C in LB solid medium. Single clones were picked and plated in kan +The strains were cultured in LB liquid culture medium at 37°C and 180 rpm for 6 hours. The bacterial solution was sent to Shanghai Biotech for sequencing. The strains with correct sequencing were preserved and stored at -80°C.

[0050] 3. Prokaryotic expression, purification and renaturation of Nb12

[0051] (1) The Nb12 strain obtained in step 2 above was spread on LB medium containing kan+ and incubated at 37°C overnight. A single colony was picked and incubated in LB liquid medium containing kan+ for 12 hours at 37°C and 180 rpm to prepare a seed solution. The culture was then expanded at a ratio of seed solution: LB liquid medium = 1:100, and IPTG with a final concentration of 1 mM was added to induce the expression of the target protein. The culture was continued at 37°C for 5 hours. After the bacterial solution was broken and centrifuged, the SDS-PAGE test results were as follows: Figure 1 As shown in Figure 1, Nb12 mainly exists in the form of inclusion bodies. GBS-Nb12 protein was purified by Ni column affinity chromatography. Figure 1 The Nb12 protein shown was purified successfully.

[0052] (2) Renaturation. Dissolve the Nb12 protein in Inclusion Body Binding Buffer (20 mL 1 M Tris-HCl (pH = 8.0), 0.68 g imidazole, 29.22 g sodium chloride, 480 g urea, dilute to 1 L with ultrapure water, adjust pH to 8.0), and then place in a dialysis bag with a molecular cutoff of 3 kD. Dialyze in the following solutions at 4 ° C for 12 h:

[0053] Dialysate A (3.125 g Tis-HCl was dissolved in 80 mL ultrapure water and then the volume was adjusted to 1 L, and the pH was adjusted to 8.0);

[0054] Dialysate B1 (3.125 g Tis-HCl, 360 g urea fully dissolved in ultrapure water, fixed to 1 L, pH adjusted to 8.0);

[0055] Dialysate B2 (3.125 g TisHC, 240 g urea fully dissolved in ultrapure water, fixed to 1 L, pH adjusted to 8.0);

[0056] Dialysate B3 (3.125 g Tris-HC, 120 g urea fully dissolved in ultrapure water, fixed to 1 L, pH adjusted to 8.0);

[0057] Dialysate B4 (3.125 g Tris-HCl, 60 g urea fully dissolved in ultrapure water, fixed to 1 L, pH adjusted to 8.0);

[0058] Dialysate C (3.125 g Tris-HCl, 0.104 g L-arginine, 100 g sucrose, 0.584 g EDTA, and 30 g urea were completely dissolved in 800 mL ultrapure water, diluted to 1 L, and the pH was adjusted to 8.0);

[0059] Dialysate D (3.125 g Tris-HC, 1.461 g NaCl, 12 g urea fully dissolved in ultrapure water, fixed to 1 L, adjusted to pH 8.0).

[0060] 4. Antigen-antibody specific binding experiment

[0061] Add the purified SIP protein to a 96-well ELISA plate (4ng / ul, coating solution is PBS solution, pH8.0, 100ul per well), and coat overnight at 4°C. Wash the ELISA plate 3 times with PBS solution T (1x PBS solution, containing 0.05% Tween 20), add blocking solution (2.5% skim milk powder) and block at room temperature for 1h. Add gradient dilutions of Nb12 and blocking solution according to the ratio of gradient dilution of Nb12: blocking solution = 1:1, and incubate at 37°C for 2h. Wash 3 times with PBS solution T, add 100ul Anti-VHH Antibody (HRP) (1:5000 diluted in blocking solution) to each well, and incubate at 37°C for 1h. Wash 3 times with PBS solution T, add 100ul TMB two-component colorimetric solution and color at 37°C in the dark for 15min, and finally add 50ul ELISA stop solution to stop color development, and measure the OD450 value with an ELISA reader. Figure 2 As shown, the NB12 nanobody has a positive reaction with the SIP protein antigen protein.

[0062] 5. Verification of the antibacterial activity of Nb12 in vivo

[0063] Healthy tilapia weighing 6±0.2 g were selected and divided into 3 groups, 5 fish in each group, and the following treatments were performed:

[0064] Control group: intraperitoneal injection of 100ul PBS solution;

[0065] Experimental group: intraperitoneal injection of 100ul of 5x10 8 cfu / ml Streptococcus agalactiae (GBS);

[0066] Treatment group: 100ul of 5x10 8 cfu / ml GBS and 100mg / kg nanobody mixture. After 24hpi, three fish were randomly selected from each group, liver tissues were sampled, tissue DNA was extracted, and real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the bacterial copy number of GBS.

[0067] The average copy number of GBS in the liver tissue of tilapia in the experimental group was 9.73x10 5 After GBS was co-incubated with Nb12, the average copy number of GBS in liver tissue decreased to 2.61x10 4 The average antibacterial rate reached 97.31%.

[0068] The present invention uses the SIP protein of tilapia-derived Streptococcus agalactiae as a target to screen specific nanoantibodies from a phage display library. The results show that the screened nanoantibody Nb12 can efficiently and specifically bind to the SIP protein, effectively inhibit the proliferation of GBS in tilapia, and has excellent antibacterial activity.

[0069] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish, characterized in that: The nanobody Nb12 comprises a heavy chain variable region VHH, wherein the VHH comprises four antibody variable region framework regions FR1, FR2, FR3, FR4 and three complementary determining clusters CDR1, CDR2, CDR3; In the nanoantibody Nb12, the amino acid sequence of FR1 is shown in SEQ ID NO.1, the amino acid sequence of FR2 is shown in SEQ ID NO.2, the amino acid sequence of FR3 is shown in SEQ ID NO.3, the amino acid sequence of FR4 is shown in SEQ ID NO.4, the amino acid sequence of CDR1 is shown in SEQ ID NO.5, the amino acid sequence of CDR2 is shown in SEQ ID NO.6, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

7.

2. The nanoantibody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish according to claim 1, characterized in that: The framework regions FR1-4 and complementary determining clusters CDR1-3 are arranged alternately in sequence, and the amino acid sequence of the heavy chain variable region VHH is shown in SEQ ID NO:

8.

3. The method for preparing the nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish according to any one of claims 1 to 2, characterized in that: The steps include: S1. Use genetic engineering technology to insert the VHH gene into the phage coat protein structural gene. As the phage reproduces, the VHH will be expressed as a fusion protein on the coat protein of the progeny phage. S2, then utilizing the affinity interaction between the antigen coated in the fixed medium and the VHH, selectively affinity panning the VHH that specifically binds to the antigen; S3. The phage-positive monoclones obtained after affinity panning are subjected to ELISA assay, and the positive clone strains are expanded and sequenced to obtain the nano-antibody Nb12 of the SIP protein.

4. The method for preparing the nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish according to claim 3, characterized in that: In the S2 step, 2-4 rounds of affinity panning are performed.

5. The method for preparing the nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish according to claim 3, characterized in that: The specific steps of affinity panning are: The coated, purified and renatured SIP protein was blocked with skimmed milk powder on the immunotube, and the VHH phage display library was added and fully incubated, and then washed 15-25 times with a washing solution to remove the phages that did not specifically bind to the SIP protein; the remaining bound phage particles were eluted with an elution solution, and then 10% AEBSF was added to terminate the elution; finally, the eluted phages were infected with the composite strain, and amplified at 35-40°C overnight after infection, and the amplified phages were precipitated and purified using a PEG / 2.5M NaCl solution, and the precipitates were collected and resuspended with a PBS solution, which were specifically bound phage particles for the next round of screening.

6. The method for preparing the nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish according to claim 5, characterized in that: The washing solution is PBS solution containing 0.05% Tween 20 (T solution).

7. The method for preparing the nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish according to claim 5, characterized in that: The eluent is a 0.25 mg / mL Trypsin solution.

8. The method for preparing the nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish according to claim 5, characterized in that: The composite strain is Escherichia coli SS320 strain and M13K07 helper phage.

9. Use of the nanoantibody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish as described in any one of claims 1 to 2 in the prevention and control of fishery diseases.

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