Preparation and application of a nanobody targeting streptococcus agalactiae sip protein

Abstract

The application discloses a kind of nanobody targeting streptococcus agalactiae SIP protein and preparation and application thereof, it is related to biotechnology field, and the nanobody that the present application is screened by nanobody phage library construction, phage screening technology, and is efficiently and specifically combined with SIP protein Nanobody, the nanobody of streptococcus agalactiae SIP protein includes VHH fragment, with the amino acid sequence shown in SEQ ID NO:1.The nanobody can be combined with streptococcus agalactiae SIP protein high specificity, and the antibody can be targeted to SIP protein by the antibody, and the application discloses a kind of nanobody targeting streptococcus agalactiae SIP protein can be applied to fish disease diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the preparation and application of a nanobody targeting the SIP protein of Streptococcus agalactiae. Background Technology

[0002] Streptococcus agalactiae, also known as group B streptococcus (GBS), is a Gram-positive streptococcus that is a zoonotic pathogen. In aquaculture, Streptococcus agalactiae can infect various freshwater and saltwater fish, particularly tilapia. It colonizes the intestines of tilapia and can penetrate intestinal epithelial cells, infecting various host tissues and causing disease. Furthermore, Streptococcus agalactiae can cross the blood-brain barrier in tilapia and enter brain tissue, damaging the nervous system and accelerating mortality, with a mortality rate reaching 80%, causing significant economic losses to the aquaculture industry. Therefore, research on the diagnosis and prevention of Streptococcus agalactiae infection is of significant social and economic benefit to ensuring the healthy development of aquaculture.

[0003] Surface immunogenic protein (SIP) is a structural product of GBS (Streptococcus agalactiae) and an immune-related protein exposed on the surface of Streptococcus agalactiae. It is highly conserved, widely present in multiple serotypes of Streptococcus agalactiae from both human and bovine origins, and is involved in bacterial adhesion and colonization. Currently, the SIP gene has been identified as one of the four highly conserved genes encoding surface proteins of Streptococcus agalactiae, possessing potential immunogenicity. Therefore, developing rapid diagnostic methods and control measures targeting SIP proteins is of great significance.

[0004] Nanobodies, currently the smallest genetically engineered antibodies, have a crystal structure with a diameter of 2.5 nm, a length of approximately 4 nm, and a molecular weight of 15 kDa (about 1 / 10 that of conventional antibodies). They are the smallest known naturally occurring antigen-binding fragments. Nanobodies possess advantages such as small molecular weight, strong tissue penetration, high tolerance, high affinity, good water solubility, easy cloning and recombination, high yield, and low cost. These superior properties have driven their application in disease diagnosis and treatment. Therefore, developing rapid diagnostic methods and control measures based on nanobodies is of great significance for ensuring the healthy development of aquaculture. Summary of the Invention

[0005] To address the above shortcomings, this invention aims to develop a highly specific nanobody targeting the SIP protein of Streptococcus agalactiae to overcome the deficiencies of traditional detection methods and control measures. This will provide technical support for the development of various platforms for the detection, analysis, and prevention of Streptococcus agalactiae infections. The specific technical solution is as follows:

[0006] A nanobody targeting the SIP protein of Streptococcus agalactiae, wherein the SIP protein nanobody contains a VHH fragment and has the amino acid sequence shown in SEQ ID NO:1, namely:

[0007] AVQLVESGGGLVQAGSSLTLACAASGSTFSSSFMGWPRQGSGKEREFVAGISMNGATTYYTDSVKGRAVQLVESGGLVQAGSSLTLACAASGSTFSSSFMGWPRQGSGKEREFVAGISMMNGATTYYTDSVKGRFTISRDNTKAAVYLQMNNLKPEDTAIYYCAVHVGHPLKFRSSDEYDNWGQCRGVRVSS.

[0008] Furthermore, this nanobody exhibits excellent binding performance and specificity to Streptococcus agalactiae SIP protein.

[0009] Furthermore, the amino acid sequence of the SIP protein is shown in SEQ ID NO.2, namely:

[0010] MKMNKKVLLTSTMAASLLSVASVQAQETDTTWTARTVSEVKADLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLTVTYDQKSHTATSMK IETPATNAAGQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMKTYSSAPALKSKEVLAQGQAVSQAAANEQVSPAPVKSITSEVPAAKEEVKPTQTSVSQ STTVSPASVAAETPAPVAKVAPVRTVAAPRVASVKVVTPKVETGASPEHVSAPAVPVTTTSTATDSKLQATEVKSVPVAQKAPTATPVAQPASTTNAVAAHPENARLQP HVAAYKEKVASTYGVNEFSTYRAGDPGDHGKGLAVDFIVGKNQALGNEVAQYSTQNMAANNISYVIWQQKFYSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFNK.

[0011] Furthermore, the method for preparing the *Streptococcus agalactiae* SIP protein is as follows:

[0012] (1) Based on the SIP gene sequence of Streptococcus agalactiae published by NCBI, sequence alignment analysis was performed, and specific amplification primers were designed. Using Streptococcus agalactiae genomic DNA as a template, the gene sequence of virulence factor was ligated into plasmid pET-28a and transformed into Escherichia coli BL21 through PCR amplification, gel recovery, double enzyme digestion, ligation and transformation steps.

[0013] (2) After the recombinant Escherichia coli was cultured to the logarithmic growth phase, the target protein was induced to be expressed by isopropyl-β-D-thiogalactoside. The expressed SIP protein was purified by nickel column to obtain high-purity SIP protein, and the purified SIP protein was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

[0014] This invention also provides a method for preparing nanobodies targeting SIP protein of Streptococcus agalactiae, comprising the following steps:

[0015] (1) Add recombinant SIP protein to an immunotube, incubate with nanobody phage display library, wash away non-specifically bound recombinant phage with PBST, elute and screen for specifically bound phage, amplify and purify the eluted phage, determine the titer, and use it for the next round of solid-phase panning.

[0016] (2) Using Streptococcus agalactiae SIP protein as the target, the selected phage library was identified by Phage-ELISA; several phage display nanobodies with good binding effect to Streptococcus agalactiae SIP protein were obtained; the selected nanobody phages were amplified and sequenced to obtain a positive nanobody, namely a nanobody targeting Streptococcus agalactiae SIP protein.

[0017] Furthermore, the elution screening specifically includes the following steps:

[0018] (1) The purified and refolded SIP protein was coated in an immunotube and coated at 4°C for 10-14 h.

[0019] (2) Wash the immunotubes three times with PBS buffer, add blocking solution and rotate to block for 2 hours;

[0020] (3) Wash the immunotubes three times with PBS buffer and add Nb immature phage display library to a final volume of 1×10⁻⁶. 12 PFU, incubated at room temperature by rotation for 1 hour;

[0021] (4) Discard the liquid in the immunotherapy tube and wash with PBST solution 18-25 times to remove unbound phages;

[0022] (5) Elute the remaining bound phage particles with 0.25 mg / mL Trypsin solution for 20-40 min;

[0023] (6) Then add 10% AEBSF to stop elution, transfer the remaining liquid in the immunoassay tube to a clean centrifuge tube, and take the phage obtained by elution and screening for titer detection.

[0024] Furthermore, the solid-phase panning method of "elution-amplification-purification" is subjected to 2-4 rounds of screening.

[0025] This invention provides a nanobody targeting the SIP protein of Streptococcus agalactiae as described above. The phage display nanobody of this invention can specifically bind to the SIP protein of Streptococcus agalactiae and can be applied to the prevention and control of fishery diseases.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1. Compared with traditional monoclonal antibodies, the nanobodies prepared by this invention have advantages such as small molecular weight, strong tissue penetration ability, high stability, and easy expression.

[0028] 2. This invention provides a method for screening nanobodies targeting the SIP protein of Streptococcus agalactiae from tilapia, thereby screening out nanobodies that bind to the SIP protein with high efficiency and specificity. These nanobodies can be used as core detection elements and drug development molecules, and applied to a platform for the detection, analysis and prevention of Streptococcus agalactiae disease. 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 This is the identification result of double enzyme digestion of the recombinant plasmid pET28a-sip in Example 1 of this invention;

[0031] Figure 2 This is the result of SIP and SDS-PAGE detection of recombinant protein in Example 1 of the present invention. Lane 1 is the empty vector of Escherichia coli BL21; lane 2 is the supernatant of bacterial culture after induction expression; lane 3 is the precipitate of bacterial culture after lysis and centrifugation.

[0032] Figure 3 This refers to the SDS-PAGE detection results of the recombinant protein SIP purification in Example 1 of this invention;

[0033] Figure 4 In Example 1 of this invention, the affinity and specificity of phage 1-50 displaying nanobodies were verified by Phage-ELISA. Detailed Implementation

[0034] 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.

[0035] Example 1

[0036] 1. Preparation of recombinant SIP protein

[0037] (1) Using *Streptococcus agalactiae* genomic DNA as a substrate, the SIP gene was amplified using primers SIP-F2 / SIP-R2. 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) by heat transformation. The cells were then plated in LB medium containing Kan+ and cultured for 10-14 h. Single colonies were picked and cultured in 1 mL of LB medium containing Kan+ at 37℃ and 180 rpm for 6 h. The plasmid was extracted and subjected to double enzyme digestion for identification. The identification results are as follows: Figure 1 As shown, the target gene SIP is approximately 1800 bp, and the pET28a-sip vector was successfully constructed.

[0038] (2) Prokaryotic expression: E. coli BL21 cells introduced with pET28a-sip were cultured in LB broth containing kan+. After IPTG induction, disruption, and centrifugation, the SDS-PAGE results of the SIP protein were analyzed. Figure 2 As shown, the Sip protein mainly exists in the form of inclusion bodies. The Sip protein was purified using Ni column affinity chromatography, and the SDS-PAGE results are shown below. Figure 3 As shown, the SIP protein is approximately 63 kDa in size, and the target protein was successfully purified.

[0039] (3) Inclusion body protein refolding: Inclusion bodies were 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, and ultrapure water, then brought to a final volume of 1 L and adjusted to pH 8.0). The solutions were then placed in dialysis bags with a molecular weight cutoff of 3 kDa and dialyzed at 4°C for 12 h in the following solutions:

[0040] Dialysis buffer A (3.125g Tis-HCl was fully dissolved in ultrapure water and brought to a final volume of 1L, then the pH was adjusted to 8.0);

[0041] 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);

[0042] 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);

[0043] 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);

[0044] 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);

[0045] 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);

[0046] 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).

[0047] 2. VHH selection targeting GBS-SIP

[0048] (1) First round of phage panning. Purified and renatured SIP protein (50 μg per tube) was coated onto immunotubes and incubated at 4°C for 10-14 h. The liquid in the 10-14 h coated immunotubes was discarded, and 2 mL of PBS buffer was added. The immunotubes were washed three times at room temperature, followed by 2 mL of blocking buffer (5% skim milk solution) and rotation blocking for 2 h. 2 mL of PBS buffer was added and washed three times. Nb immature phage display library was added to a concentration of 1 × 10¹² PFU and incubated at room temperature for 1 h. The liquid in the immunotubes was discarded, and 2 mL of PBST (1x PBS, containing 0.05% Tween 20) was added and washed 20 times to remove unbound phages. 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 the elution. The remaining liquid in the immunoassay tube was transferred to a clean 1.5 mL centrifuge tube, which contained the phages from the first round of panning. 10 μL of the panned phages was then used for titer analysis.

[0049] (2) Amplification of eluted phages. *Escherichia coli* SS320 strain was streaked onto 2xYT solid medium (Tet-resistant) and incubated at 37°C for 10–14 h. Single colonies were picked from the streaks and transferred to 5 ml of 2xYT medium containing 10 μg / mL Tet, and incubated at 37°C for 10–14 h. 250 μL of the 10–14 h culture was transferred to 5 mL of 2xYT liquid medium containing Tet resistance, and incubated at 37°C and 250 rpm for 45 min. 500 μL of the first round of panning phages was added, and the culture was continued at 37°C and 220 rpm for 30 min. The culture was then spread onto 245 mm square agarose plates containing 100 μg / mL Amp and 2% glucose, and incubated at 37°C for 10–14 h. Add 6 mL of 10 μg / mL Tet-containing 2x YT liquid medium to elute all single colonies on the plate to construct a phage nanobody library. Transfer the library to 100 mL of Tet-resistant 2x YT liquid medium and incubate at 37°C until the logarithmic growth phase. Add M13K07 helper phage to make the ratio of E. coli to helper phage 1:20. Continue incubation at 37°C and 220 rpm for 30 min. Then add Kan to a final concentration of 50 μg / mL and 0.2 μM IPTG, and incubate at 37°C and 250 rpm for 10–14 h.

[0050] (3) Phage purification. Centrifuge the bacterial culture (10-14 h) at 4000 rpm and 4°C for 10 min. Collect the supernatant, add 10 mL of PEG / 2.5 M NaCl, mix well, and incubate on ice for 30 min. Centrifuge at 4000 rpm and 4°C for 20 min, discard the supernatant, resuspend in 1 mL of PBS, add 250 μL of PEG / 2.5 M NaCl, and incubate on ice for 10 min. Centrifuge at 12000 rpm and 4°C for 20 min, discard the supernatant, resuspend in 1 mL of PBS, centrifuge at 12000 rpm and 4°C for 2 min, and collect the supernatant. This is the first round of phage screening sub-library; use 10 μL for titer determination.

[0051] (4) Second and third rounds of phage panning, amplification and purification. 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 unchanged. The phage titer was determined after each round of screening to obtain a phage sub-library.

[0052] 3. Monoclonal ELISA assay

[0053] Purified SIP protein was added to a 96-well ELISA plate (4 ng / µl, PBS, pH 8.0, 100 µl per well) and coated at 4°C for 10–14 h. The plate was washed three times with PBST, and blocking buffer (2.5% skim milk solution) was added and incubated 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 the plate was 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 the plate was 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 color development. The OD450 value was measured using a microplate reader. A positive reaction was considered to be a ratio of absorbance of the assay well to absorbance of the blank well > 2.1. Positive clone strains were amplified and sent to Shanghai Sangon Biotech for sequencing analysis. By comparing the sequenced samples, an anti-SIP nanobody was obtained.

[0054] This invention uses the SIP protein of Streptococcus agalactiae from fish as a target to screen specific nanobodies from a phage display library. The results show that the screened nanobodies that bind to the SIP protein with high efficiency and specificity can be used as core detection elements and drug development molecules, and applied to a platform for the detection, analysis and prevention of Streptococcus agalactiae disease.

[0055] 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 targeting a Streptococcus agalactiae SIP protein, characterized in that, The Nanobody targeting the SIP protein of Streptococcus agalactiae comprises a VHH fragment having the amino acid sequence shown in SEQ ID NO:

1.

2. The Nanobody targeting a Streptococcus agalactiae SIP protein according to claim 1, characterized in that, The amino acid sequence of the SIP protein is shown in SEQ ID NO.

2.

3. Use of a Nanobody targeting the SIP protein of Streptococcus agalactiae according to claim 1 or 2 for the manufacture of a diagnostic test for Streptococcus agalactiae disease.

Images