Preparation and application of nano antibody targeting streptococcus agalactiae SIP (Session Initiation Protocol) protein

Abstract

The invention discloses preparation and application of a nano antibody targeting streptococcus agalactiae SIP protein, and relates to the technical field of biology, the nano antibody efficiently and specifically bound with the SIP protein is screened out through nano antibody phage library construction and phage screening technologies, the nano antibody of the streptococcus agalactiae SIP protein comprises a VHH fragment, and the VHH fragment is a VHH fragment. The gene has an amino acid sequence as shown in SEQ ID NO: 1. The nanometer antibody can be combined with streptococcus agalactiae SIP protein in a high-specificity mode, the antibody can act on the SIP protein in a targeting mode, and the nanometer antibody targeting the streptococcus agalactiae SIP protein can be applied to diagnosis and treatment of fish diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the preparation and application of a nano antibody targeting a Streptococcus agalactiae SIP protein. Background Art

[0002] Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a Gram-positive streptococcus and a pathogenic bacterium that is common to humans, livestock and aquatic animals. In aquaculture, Streptococcus agalactiae can infect a variety of freshwater fish, especially tilapia. Streptococcus agalactiae can colonize in the intestines of tilapia, and can pass through intestinal epithelial cells to infect various tissues of the host, causing fish disease. In addition, Streptococcus agalactiae can break through the blood-brain barrier of tilapia and enter the brain tissue, thereby damaging the nervous system, causing tilapia to die faster. The mortality rate after infection can reach 80%, causing huge economic losses to the aquaculture industry. Therefore, conducting research on the diagnosis and prevention of Streptococcus agalactiae disease has important social and economic benefits for ensuring the healthy development of the aquaculture industry.

[0003] Surface immunogenic protein (SIP) is a structural product of GBS. It is an immune-related protein exposed on the surface of Streptococcus agalactiae. It is highly conserved and widely present in multiple serotypes of Streptococcus agalactiae strains of human and bovine origin, and is related to bacterial adhesion and colonization. At present, the SIP gene has been confirmed to be one of the four highly conserved surface protein genes encoding Streptococcus agalactiae, which has potential immunogenicity. Therefore, it is of great significance to develop rapid diagnostic methods and prevention and control measures targeting SIP protein.

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

[0005] In view of the above shortcomings, the present invention aims to solve the shortcomings of traditional detection methods and prevention and control measures, and aims to develop a highly specific nanoantibody targeting the SIP protein of Streptococcus agalactiae, so as to provide certain technical support for the development of various detection, analysis and prevention and control platforms of Streptococcus agalactiae disease. The specific technical scheme is as follows:

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

[0007] AVQLVESGGGLVQAGSSLTLACAASGSTFSSSFMGWPRQGSGKEREFVAGISMNGATTYYTDSVKGRAVQLVESGGLVQAGSSLTLACAASGSTFSSSFMGWPRQGSGKEREFVAGISMMNGATTYYTDSVKGRFTISRDNTKAAVYLQMNNLKPEDTAIYYCAVHVGHPLKFRSSDEYDNWGQCRGVRVSS.

[0008] Furthermore, the nanoantibody has excellent binding properties and specificity with the SIP protein of Streptococcus agalactiae.

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

[0010] MKMNKKVLLTSTMAASLLSVASVQAQETDTTWTARTVSEVKADLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLTVTYDQKSHTATSMK IETPATNAAGQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMKTYSSAPALKSKEVLAQGQAVSQAAANEQVSPAPVKSITSEVPAAKEEVKPTQTSVSQ STTVSPASVAAETPAPVAKVAPVRTVAAPRVASVKVVTPKVETGASPEHVSAPAVPVTTTSTATDSKLQATEVKSVPVAQKAPTATPVAQPASTTNAVAAHPENARLQP HVAAYKEKVASTYGVNEFSTYRAGDPGDHGKGLAVDFIVGKNQALGNEVAQYSTQNMAANNISYVIWQQKFYSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFNK.

[0011] Further, the preparation method of the Streptococcus agalactiae SIP protein is as follows:

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

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

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

[0015] (1) Add the recombinant SIP protein to the immunotube, incubate with the nanobody phage display library, wash away the non-specifically bound recombinant phage with PBST, elute and screen the 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 the SIP protein of Streptococcus agalactiae as the target, the selected phage library was identified by Phage-ELISA; several phage-displayed nanoantibodies that specifically bind to the SIP protein of Streptococcus agalactiae with better effects were obtained, and the selected nanoantibody phages were amplified, sequenced and compared to obtain a positive nanoantibody, i.e., a nanoantibody targeting the SIP protein of Streptococcus agalactiae.

[0017] Further, the elution screening specifically comprises the following steps:

[0018] (1) Coat the purified and renatured SIP protein in an immunotube at 4°C for 10-14 hours;

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

[0020] (3) Wash the immunotube three times with PBS buffer and add the Nb naive phage display library to 1×10 12 PFU, incubate with rotation at room temperature for 1 h;

[0021] (4) Discard the liquid in the immunotube 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, 10% AEBSF was added to terminate the elution, and the remaining liquid in the immunotube was transferred to a clean centrifuge tube. The phages obtained by elution and screening were taken for titer detection.

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

[0025] The present invention provides a nano antibody targeting the SIP protein of Streptococcus agalactiae as described above. The phage-displayed nano antibody of the present invention can specifically bind to the SIP protein of Streptococcus agalactiae and is applied to the prevention and control of fishery diseases.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

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

[0028] 2. The present invention provides a method for screening nanoantibodies targeting the SIP protein of tilapia-derived Streptococcus agalactiae, and screens out nanoantibodies that efficiently and specifically bind to the SIP protein. The nanoantibodies can be used as core detection elements and drug development molecules and applied to the detection, analysis and prevention and control platform of Streptococcus agalactiae disease. 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 It is the result of double enzyme digestion identification of the recombinant plasmid pET28a-sip in Example 1 of the present invention;

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

[0032] Figure 3 The results of SDS-PAGE analysis of the recombinant protein SIP purification in Example 1 of the present invention are as follows;

[0033] Figure 4 In Example 1 of the present invention, the affinity and specificity of the 1-50 phage-displayed nanoantibody were verified by Phage-ELISA. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1

[0036] 1. Preparation of recombinant SIP protein

[0037] (1) Using the genomic DNA of Streptococcus agalactiae as the substrate, the SIP gene was amplified with primers SIP-F2 / SIP-R2 respectively. The PCR product was detected by 1% agarose gel electrophoresis, connected to the pET28a plasmid through the restriction endonuclease sites EcoRI and Hind III, and transferred into the competent Escherichia coli BL21 (DE3) cells by thermal transformation, and then spread on LB medium containing kan+ for 10-14 hours. Pick a single clone colony into 1mL LB medium containing kan+, culture at 37°C, 180rpm for 6 hours, extract the plasmid for double enzyme digestion identification, and the identification results are as follows: Figure 1 As shown, the target gene SIP is about 1800 bp, and the pET28a-sip vector is successfully constructed.

[0038] (2) Prokaryotic expression: E. coli BL21 after the introduction of pET28a-sip was cultured in LB liquid medium containing kan+. After IPTG induction, fragmentation, centrifugation and other steps, the SDS-PAGE detection results of SIP protein Figure 2 As shown in Figure 2, Sip protein mainly exists in the form of inclusion bodies. Sip protein was purified by Ni column affinity chromatography, and the results of SDS-PAGE detection are shown in Figure 2. Figure 3 As shown, the size of the SIP protein was approximately 63 kDa, and the target protein was purified successfully.

[0039] (3) Inclusion body protein refolding: The 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 the volume was adjusted to 1 L with ultrapure water after being fully dissolved, and the pH was adjusted to 8.0). Then, the inclusion bodies were placed in a dialysis bag with a molecular cutoff of 3 kD and dialyzed in the following solutions at 4 °C for 12 h:

[0040] Dialysate A (3.125 g Tis-HCl was fully dissolved in ultrapure water and then diluted to 1 L, and the pH was adjusted to 8.0);

[0041] Dialysate B1 (3.125 g Tis-HCl, 360 g urea fully dissolved in ultrapure water, fixed to 1 L, pH adjusted to 8.0);

[0042] Dialysate B2 (3.125 g TisHC, 240 g urea fully dissolved in ultrapure water, fixed to 1 L, pH adjusted to 8.0);

[0043] Dialysate B3 (3.125 g Tris-HC, 120 g urea fully dissolved in ultrapure water, fixed to 1 L, pH adjusted to 8.0);

[0044] Dialysate B4 (3.125 g Tris-HCl, 60 g urea fully dissolved in ultrapure water, fixed to 1 L, pH adjusted to 8.0);

[0045] 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);

[0046] 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).

[0047] 2. VHH Selection Targeting GBS-SIP

[0048] (1) The first round of phage panning. The purified renatured SIP protein (50ug per tube) was coated on the immunotubes at 4°C for 10-14h. The liquid in the immunotubes coated for 10-14h was discarded, 2mL PBS buffer was added, and the immunotubes were washed 3 times at room temperature. Then 2mL blocking solution (5% skim milk powder solution) was added and rotated for blocking for 2h. 2mL PBS buffer was added for washing 3 times, and the Nb naive phage display library was added to 1×1012PFU, and the tubes were rotated for incubation at room temperature for 1h. The liquid in the immunotubes was discarded, and 2mL PBST (1x PBS, containing 0.05% Tween 20) was added and washed 20 times to remove unbound phages. Use 1mL 0.25mg / mL Trypsin solution to elute the remaining bound phage particles for 30 minutes, then add 10μL 10% AEBSF to terminate the elution. Transfer the remaining liquid in the immunotube to a clean 1.5mL centrifuge tube to obtain the phages from the first round of panning. Take 10uL of the panned phages for titer detection.

[0049] (2) Amplification of eluted phages. E. coli SS320 strain was streaked with single colonies on 2xYT solid medium (Tet resistance) and cultured at 37°C for 10-14h. Single colonies were picked from the single colony plate and transferred to 5ml of 2x YT medium containing 10ug / mL Tet and cultured at 37°C for 10-14h. 250uL of the 10-14h culture solution was transferred to 5mL of 2x YT liquid medium containing Tet resistance, cultured at 37°C, 250rpm for 45min, 500uL of the first round of panning phage was added, and cultured at 37°C, 220rpm for 30min. The bacterial solution was spread on a 245mm square culture medium plate containing 2% agarose with 100ug / mL Amp and 2% glucose, and cultured at 37°C for 10-14h. 6 mL of 10 ug / mL Tet in 2x YT liquid medium was added to elute all single colonies on the plate to constitute the phage nanoantibody library, which was then transferred to 100 ml of Tet-resistant 2x YT liquid medium, and the bacterial solution was cultured at 37°C to the logarithmic growth phase. M13K07 helper phage was added to make the number of Escherichia coli: number of helper phage = 1:20. After continuing to culture at 37°C and 220 rpm for 30 min, Kan with a final concentration of 50 ug / mL and IPTG of 0.2 uM were added, and cultured at 37°C and 250 rpm for 10-14 h.

[0050] (3) Purification of phages. Take the bacterial solution cultured for 10-14 hours, centrifuge at 4000 rpm, 4°C for 10 minutes. Take the supernatant, add 10 mL PEG / 2.5 M NaCl, mix well and let stand on ice for 30 minutes. Centrifuge at 4000 rpm, 4°C for 20 minutes, discard the supernatant, resuspend in 1 mL PBS, add 250 ul PEG / 2.5 M NaCl, let stand on ice for 10 minutes. Centrifuge at 12000 rpm, 4°C for 20 minutes, discard the supernatant, resuspend in 1 mL PBS, centrifuge at 12000 rpm, 4°C for 2 minutes, collect the supernatant, which is the first round of phage screening sub-library, and take 10 uL for titer determination.

[0051] (4) Second and third rounds of phage selection, amplification and purification. The remaining steps remain unchanged except that the phage added to the immunotube is changed to the phage obtained by purification and amplification after the previous round of screening. The phage titer is measured after each round of screening to obtain the phage sub-library.

[0052] 3. Monoclonal ELISA Assay

[0053] The purified SIP protein was added to a 96-well ELISA plate (4ng / ul, the coating solution was PBS, pH8.0, 100ul per well), and coated at 4℃ for 10-14h. The ELISA plate was washed 3 times with PBST, and blocked with blocking solution (2.5% skim milk powder solution) at room temperature for 1h. The phage sub-library obtained after the third round of panning amplification: blocking solution = 1:9 was added to the phage library and blocking solution, and incubated at 37℃ for 2h. Then 100ul Anti-VHH Antibody (HRP) was diluted 1:5000 in the blocking solution to each well, and incubated at 37℃ for 1h. 100ul TMB two-component color development solution was added to develop color at 37℃ in the dark for 15min, and finally 50ul ELISA stop solution was added to stop the color development, and the OD450 value was measured with an ELISA reader. The absorbance value of the measured well / the absorbance value of the blank well>2.1 was considered a positive reaction. The positive clone strains were cultured and sent to Shanghai Bioengineering for sequencing analysis. By comparing the sequence, an anti-SIP nanobody was obtained.

[0054] The present invention uses the fish-derived Streptococcus agalactiae SIP protein as a target to screen specific nanoantibodies from a phage display library. The results show that the screened nanoantibodies that efficiently and specifically bind to the SIP protein can be used as core detection elements and drug development molecules, and applied to the detection, analysis and prevention and control platform of Streptococcus agalactiae disease.

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

1.

2. The nanobody targeting the SIP protein of Streptococcus agalactiae according to claim 1, characterized in that The nano antibody has excellent binding performance and specificity with the SIP protein of Streptococcus agalactiae.

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

2.

4. The nanobody targeting the SIP protein of Streptococcus agalactiae according to claim 1, characterized in that The preparation method of the Streptococcus agalactiae SIP protein is as follows: (1) According to the SIP gene sequence of Streptococcus agalactiae published by NCBI, sequence alignment analysis was performed, and specific amplification primers were designed. The genomic DNA of Streptococcus agalactiae was used as a template. After PCR amplification, gel recovery, double enzyme digestion, ligation and transformation steps, the gene sequence of the virulence factor was connected to the plasmid pET-28a and transformed into Escherichia coli BL21 to obtain recombinant Escherichia coli; (2) After the recombinant Escherichia coli was cultured to the logarithmic growth phase, isopropyl-β-D-thiogalactoside was used to induce the expression of the target protein. The expressed SIP protein was purified using a nickel column to obtain a high-purity SIP protein, and the purified SIP protein was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

5. The method for preparing a nanobody targeting the SIP protein of Streptococcus agalactiae according to claim 1, characterized in that: The steps include: (1) Add the recombinant SIP protein to the immunotube, incubate with the nanobody phage display library, wash away the non-specifically bound phages with PBST, elute and screen the specifically bound phages, amplify and purify the eluted phages, determine the titer, and use them for the next round of solid phase panning; (2) The selected phage library is identified by Phage-ELISA, using the Streptococcus agalactiae SIP protein as the target; several recombinant phage-displayed nanoantibodies with better specific binding to the Streptococcus agalactiae SIP protein are obtained, and the selected nanoantibody phages are amplified, sequenced and compared to obtain a positive nanoantibody, i.e., a nanoantibody targeting the Streptococcus agalactiae SIP protein.

6. The method for preparing a nanobody targeting the SIP protein of Streptococcus agalactiae according to claim 5, characterized in that: The elution screening specifically comprises the following steps: (1) Coat the purified and renatured SIP protein in an immunotube at 4°C for 10-14 hours; (2) Wash the immunotube three times with PBS buffer, add blocking solution and rotate to block for 2 h; (3) Wash the immunotube three times with PBS buffer and add the nanobody phage display library to 1×10 12 pfu, incubated with rotation at room temperature for 1 h; (4) Discard the liquid in the immunotube and wash with PBST solution 18-25 times to remove unbound phages; (5) Elute the remaining bound phage particles with 0.25 mg / mL Trypsin solution for 20-40 min; (6) Then, 10% AEBSF was added to terminate the elution, and the remaining liquid in the immunotube was transferred to a clean centrifuge tube. The phages obtained by elution and screening were taken for titer detection.

7. The method for preparing a nanobody targeting the SIP protein of Streptococcus agalactiae according to claim 5, characterized in that: The solid phase panning was performed for 2-4 rounds of screening.

8. Use of the nanobody targeting the SIP protein of Streptococcus agalactiae according to any one of claims 1 to 7 in the prevention and control of fishery diseases.

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