Preparation and application of a nanobody Nb12 targeting SIP protein of Streptococcus agalactiae in fish

Abstract

This invention discloses the preparation and application of a nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish, relating to the field of fisheries disease control technology. The nanobody of this invention, screened using phage display technology, targets the SIP protein of Streptococcus agalactiae from tilapia and possesses a heavy chain variable region (VHH). The VHH includes four intracellular backbone regions (FR1, FR2, FR3, FR4) and three complementarity determinants (CDR1, CDR2, CDR3). The nanobody Nb12 provided by this invention exhibits high affinity for the SIP protein of Streptococcus agalactiae, allowing it to target the SIP protein effectively and inhibiting GBS proliferation in tilapia, demonstrating excellent antibacterial activity and its application in the prevention and control of diseases in tilapia.

Description

Technical Field

[0001] This invention relates to the field of fishery disease control technology, and in particular to a nanobody Nb12 that targets the SIP protein of Streptococcus agalactiae in fish and its application. Background Technology

[0002] Tilapia (Oreochromis sp.) is one of the most important aquaculture species recommended globally by the Food and Agriculture Organization of the United Nations (FAO). In 2023, my country's tilapia production 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 major tilapia farming areas in my country. Its devastating impact has led to it being dubbed the "H1N1" of tilapia farming, causing huge economic losses. Currently, antibiotics remain the main method for controlling Streptococcus agalactiae disease in aquaculture environments. However, the long-term use and abuse of antibiotics have not only led to severe drug resistance in Streptococcus agalactiae but have also caused a series of problems such as environmental pollution and food safety.

[0003] In the development of streptococcal disease in tilapia, *Streptococcus agalactiae* virulence factors play a crucial role in invasion and lysis of host cells. These virulence factors are numerous and can be broadly classified into two categories: structural products and secretory products. Structural products are generally associated with bacterial adhesion and colonization, while secretory products are often involved in invasion and damage to host cells and tissues. Among these, surface immunogenic protein (SIP) is a structural product of *GBS*, located on the bacterial surface, and participates in the adhesion and colonization of host cells and tissues. All serotypes of *GBS* express SIP proteins, and the SIP gene is highly conserved in *GBS*, possessing potential immunogenicity and serving as an important candidate protein for *Streptococcus agalactiae* vaccine antigens.

[0004] In the 1990s, Hamers et al. discovered that camel serum contains naturally occurring heavy-chain antibodies (HCAbs) that lack the light chain portion and contain only the heavy chain portion. Cloning the variable region gene of HCAbs in camels yields a single-domain antibody (VHH) composed solely of the heavy chain variable region, also known as nanobodies (Nbs). The single domain of VHH possesses excellent properties such as small molecular weight, strong tissue penetration, high tolerance, high affinity, good water solubility, easy cloning and recombination, high yield, and low cost, promoting its application in disease diagnosis and treatment.

[0005] Therefore, it is urgent to develop a novel green control method for Streptococcus agalactiae by utilizing the superior characteristics of VHH. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a nanobody, Nb12, targeting the SIP protein of Streptococcus agalactiae in fish. This nanobody can be used to prepare neutralizing antibodies that inhibit Streptococcus agalactiae infection in fish, solving the current problem of a lack of effective neutralizing antibodies for the prevention and control of Streptococcus agalactiae disease in fish. The specific technical solution 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 includes four antibody variable region backbone regions FR1, FR2, FR3, and FR4 and three complementarity determinants CDR1, CDR2, and CDR3, as follows:

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

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

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

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

[0012] The amino acid sequence of CDR1 is shown in SEQ ID NO.5, namely 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, namely AASRSDYDPLFGS;

[0015] The backbone regions FR1-4 and complementarity determinants CDR1-3 are arranged in an alternating sequence, and the amino acid sequence of the heavy chain variable region VHH is shown in SEQ ID NO:8, namely:

[0016] AVQLVESGGGMVQAGGSLKLSCVASMGWFRQAQGKEREIIAGYYQDSVKDRFTIFR DNAKNTVFLQMNSLRPEDTGVYYCWGQGTQVTVSSGRTFSTLGISWTGQATAASRSDYD PLFGS.

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

[0018] S1. Using genetic engineering technology, the gene encoding VHH is inserted into the phage capsid protein structural gene. As the phage multiplies, VHH will be expressed as a fusion protein on the capsid protein of the progeny phage.

[0019] S2. Then, by utilizing the affinity between the antigen coated in the immobilization medium and VHH, the VHH that specifically binds to the antigen is selectively affinity-selected.

[0020] S3. After 2-4 rounds of affinity panning, the phage-positive monoclonal samples obtained are subjected to ELISA assay. The positive clone strains are then amplified and sequenced to obtain the SIP protein nanobody Nb12.

[0021] Furthermore, the specific steps of the affinity screening are as follows:

[0022] After coating and renaturing, the SIP protein was blocked in skim milk powder on an immunoassay tube, and a VHH phage display library was added. After incubation, the tube was washed 15-25 times with washing buffer to remove phages that did not specifically bind to the SIP protein. The remaining bound phage particles were eluted with elution buffer, and then elution was stopped by adding 10% AEBSF. Finally, the eluted phages were used to infect a composite strain, and the mixture was amplified overnight at 35-40°C. The amplified phages were purified by precipitation using PEG / 2.5M NaCl solution, and the precipitate was collected and resuspended in PBS solution. These were the specifically bound phage particles, which were used in 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 nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish. The nanobody Nb12 of the present invention can specifically bind to the SIP protein of Streptococcus agalactiae and can be used to prepare neutralizing antibodies that inhibit Streptococcus agalactiae infection in fish, as well as for the prevention and control of related fishery diseases.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] The nanobody Nb12 prepared by this invention can target the SIP protein of Streptococcus agalactiae in tilapia with high affinity, providing a foundation for the development of a new generation of green antibacterial agents and having important application value. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The following are the SDS-PAGE results of the expression and purification of recombinant protein Nb12 in Example 1 of this invention; lane 1 is the empty vector of Escherichia coli BL21; lane 2 is the whole Escherichia coli after induction of expression; lane 3 is the supernatant of the bacterial culture after induction of expression; lane 4 is the precipitate of the bacterial culture after induction of expression; lane P is the purified Nb12.

[0031] Figure 2 The results show the affinity detection of the nanobody Nb12 and the target protein SIP in Example 1 of this invention. Detailed Implementation

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

[0033] Example 1

[0034] 1. VHH selection targeting GBS-SIP

[0035] (1) First round of screening. 50 μg of purified and renatured SIP protein (prepared and preserved in the inventor's laboratory) was coated onto immunotubes and incubated overnight at 4°C. After blocking with 2 mL of 5% (w / v) skim milk powder, 100 μL of immature phage display library (Kangti Life, Shenzhen, China) was added and incubated at 37°C for 1 hour. Then, unbound phages were removed by washing 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, followed by the addition of 10 μL of 10% AEBSF to stop elution. Finally, the eluted phage was added to infect Escherichia coli SS320 strain, and after infection with M13K07 helper phage, it was amplified overnight at 37°C. The amplified phage was purified by precipitation using PEG / 2.5M NaCl solution, the precipitate was collected and resuspended in PBS solution, which were then used as 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. Except for the phages added to the immunotherapy tubes, which were all obtained by purification and amplification after the previous round of screening, the other steps remained the same, and the phage titer was determined after each round of screening.

[0037] (3) Monoclonal ELISA assay. Purified SIP protein was added to a 96-well microplate (4 ng / µl, coating buffer: PBS solution, pH 8.0, 100 µl per well), and coated overnight at 4°C. The microplate was washed three times with PBS solution T, and blocking buffer (2.5% skim milk powder) was added and blocked at room temperature for 1 h. The phage library obtained after the third round of panning and amplification was added to the blocking buffer at a ratio of 1:9, and incubated at 37°C for 2 h. Then, 100 µl of Anti-VHH Antibody (HRP) diluted 1:5000 in the blocking buffer was added to each well, and incubated at 37°C for 1 h. 100 µl of TMB two-component chromogenic solution was added, and the plate was incubated at 37°C in the dark for 15 min. Finally, 50 µl of ELISA stop solution was added to stop the chromogenic reaction, and the OD450 value was measured using a microplate reader. Positive clone strains were amplified and sent to Shanghai Sangon Biotech for sequencing analysis. By comparing the sequenced sequences, the anti-SIP nanobody Nb12 was obtained.

[0038] 2. Construction and identification of the 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 based on the VHH gene sequence as shown in Table 1:

[0040] Table 1. Primers for VHH amplification

[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 procedure is shown in Table 3:

[0047] Table 3 PCR reaction procedure

[0048]

[0049] The PCR products were detected by 1% agarose gel electrophoresis. The pET28a plasmid was ligated to the restriction endonuclease sites EcoRI and HindIII, and then transformed into E. coli competent cells BL21(DE3) via heat transformation. The transformed plasmid was then plated onto a substrate containing kan... + Incubate overnight at 37°C in LB solid medium. Pick single colonies and culture in a solution containing Kans. +The bacterial culture was incubated in LB liquid medium at 37°C and 180 rpm for 6 hours. The bacterial culture was then sent to Shanghai Sangon Biotech for sequencing. The correctly sequenced strains were preserved and stored at -80°C.

[0050] 3. Nb12 prokaryotic expression, purification and refolding

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

[0052] (2) Renaturation. Nb12 protein was dissolved 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, dissolved in ultrapure water and brought to a final volume of 1 L, pH adjusted to 8.0). The solution was then placed in a dialysis bag with a molecular weight cutoff of 3 kDa and dialyzed at 4°C for 12 h in the following solutions:

[0053] Dialysis buffer A (3.125g Tis-HCl dissolved in 80mL ultrapure water, then brought to a final volume of 1L and adjusted to pH 8.0);

[0054] Dialysis buffer B1 (3.125g Tis-HCl, 360g urea dissolved in ultrapure water, brought to a final volume of 1L, and pH adjusted to 8.0);

[0055] Dialysis buffer B2 (3.125g TisHC, 240g urea dissolved in ultrapure water, brought to a final volume of 1L, and pH adjusted to 8.0);

[0056] Dialysis buffer B3 (3.125g Tris-HC, 120g urea dissolved in ultrapure water, brought to a final volume of 1L, and pH adjusted to 8.0);

[0057] Dialysis buffer B4 (3.125g Tris-HCl, 60g urea dissolved in ultrapure water, brought to a final volume of 1L, and pH adjusted to 8.0);

[0058] Dialysis buffer C (3.125g Tris-HCl, 0.104g L-arginine, 100g sucrose, 0.584g EDTA, 30g urea completely dissolved in 800mL ultrapure water, brought to a final volume of 1L, and pH adjusted to 8.0);

[0059] Dialysis solution D (3.125g Tris-HC, 1.461g NaCl, 12g urea dissolved in ultrapure water, brought to a final volume of 1L, and pH adjusted to 8.0).

[0060] 4. Antigen-antibody specific binding assay

[0061] The purified SIP protein was added to a 96-well ELISA plate (4 ng / µl, coating buffer: PBS solution, pH 8.0, 100 µl per well) and incubated overnight at 4°C. The plate was washed three times with PBS solution T (1x PBS solution containing 0.05% Tween 20), and blocking buffer (2.5% skim milk powder) was added, with the plate blocked at room temperature for 1 h. A serially diluted Nb12 and blocking buffer were added at a 1:1 ratio, and the plate was incubated at 37°C for 2 h. The plate was washed three times with PBS solution T, and 100 µl of Anti-VHH Antibody (HRP) (1:5000 diluted in blocking buffer) was added to each well, with the plate incubated at 37°C for 1 h. The plate was washed three times with PBS solution T, and 100 µl of TMB two-component chromogenic solution was added, with the plate incubated at 37°C in the dark for 15 min. Finally, 50 µl of ELISA stop solution was added to stop the chromogenic process, and the OD450 value was measured using a microplate reader. Figure 2 As shown, the NB12 nanobody showed a positive reaction with the SIP protein antigen.

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

[0063] Healthy tilapia weighing 6±0.2g were selected and divided into 3 groups of 5 fish each. The following treatments were performed:

[0064] Control group: Intraperitoneal injection of 100 μL PBS solution;

[0065] Experimental group: Intraperitoneal injection of 100 μL at a concentration of 5 x 10⁻⁶ 8 cfu / ml agalactia-free streptococci (GBS);

[0066] Treatment group: Intraperitoneal injection of 100 μL of 5x10⁻¹⁰ g of styraxylene (SCI) co-incubated at 28°C for 30 min. 8 A mixture of cfu / ml GBS and 100 mg / kg nanobody was prepared. After 24 hpi, three fish were randomly selected from each group, and liver tissue samples were taken to extract tissue DNA. The bacterial copy number of GBS was detected by real-time quantitative PCR (RT-qPCR).

[0067] The average copy number of GBS in the liver tissue of tilapia in the experimental group was 9.73 x 10⁻⁶. 5 After co-incubating GBS with Nb12, the average GBS copy number in liver tissue decreased to 2.61 x 10⁻⁶. 4 The average antibacterial rate reached 97.31%.

[0068] This invention screens specific nanobodies from a phage display library using the SIP protein of Streptococcus agalactiae derived from tilapia as a target. The results show that the screened nanobodies Nb12 can bind to the SIP protein efficiently and specifically, and effectively inhibit the proliferation of GBS in tilapia, exhibiting excellent antibacterial activity.

[0069] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined 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 contains a heavy chain variable region VHH, which includes four antibody variable region backbone regions FR1, FR2, FR3, and FR4 and three complementarity determinants CDR1, CDR2, and CDR3. In the nanobody 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 nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish according to claim 1, characterized in that, The backbone regions FR1-4 and complementary determinants CDR1-3 are arranged in an alternating sequence, and the amino acid sequence of the heavy chain variable region VHH is shown in SEQ ID NO:

8.

3. The use of the nanobody Nb12 targeting the SIP protein of Streptococcus agalactiae in fish as described in any one of claims 1 or 2 in the preparation of a neutralizing antibody that inhibits Streptococcus agalactiae infection in fish.

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