Nano antibody for resisting vibrio parahaemolyticus and vibrio alginolyticus as well as preparation method and application thereof

Abstract

The invention discloses a nano antibody for resisting vibrio parahaemolyticus and vibrio alginolyticus as well as a preparation method and application of the nano antibody. The nano antibody for resisting vibrio parahaemolyticus and vibrio alginolyticus is characterized in that a VHH sequence is formed by a framework region, a complementarity determining region and a connecting peptide for connecting the framework region and the complementarity determining region. Nucleotide for coding an amino acid sequence of the nano antibody for specifically recognizing vibrio parahaemolyticus and vibrio alginolyticus is provided, and corresponding protein or polypeptide can be obtained through expression of the provided nucleotide sequence through a proper expression system. The preparation method comprises the following steps: designing a specific primer, obtaining cDNA (complementary deoxyribonucleic acid) of a striped bamboo shark source through PCR (polymerase chain reaction), and amplifying in a library; and cloning the gene segment of the obtained PCR product to an expression vector pPICZ alpha A, transforming the plasmid to a host cell, and carrying out induced expression and transformation on the plasmid. The nano antibody obtained by the invention has strong binding capacity and specificity to vibrio parahaemolyticus and vibrio alginolyticus.

Description

Technical Field

[0001] The present invention relates to anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibodies, preparation methods and applications thereof, and belongs to the field of nanoantibody technology and genetic engineering antibodies, and in particular to anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibodies with strong specificity, high affinity and strong stability, preparation methods and applications thereof. Background Art

[0002] Vibrionaceae bacteria are Gram-negative bacteria in the microbial flora of coastal estuaries. The bacteria are short rod-shaped, straight or curved, S-shaped or spiral-shaped. Most Vibrio have flagella, mostly single-ended polar flagella, but there are also cases where multiple flagella can be seen at one end. Vibrio are widely distributed in brackish water, estuaries, and ocean water and sediments.

[0003] Vibrio species reported at home and abroad mainly include V.alginolyticus, Vibrio parahaemolyticus, Aeromonas hydrophiva, V.harveyi, V.anguillarium, etc. Vibrio can cause farmed animals to eat less or stop eating, have decreased motor activity, abnormal behavior, and show red legs, black spots on the body shell, turbid muscles, empty stomach, sepsis and other symptoms. At present, the means of preventing and controlling aquatic pathogens mainly include antibiotics, microorganisms, Chinese herbal medicines and immunization methods. However, these methods have obvious defects, such as antibiotics are easy to remain, long-term use is easy to produce drug resistance, and most of them are listed as banned drugs; the preparation process of vaccines for immunization prevention and control is complicated, the cost is high, and the immunization effect is unstable; microbial preparations such as bacteriophages face problems such as resistance and biosafety; the results of antibacterial tests of Chinese herbal medicines are less reproducible.

[0004] Traditional IgG has a Y-shaped structure, consisting of 2 heavy chains and 2 light chains. In 1993, Hamers-Casterman C et al. found a special antibody with a size of 100 kDa that lacked light chains in the serum of dromedary camels, and called it single-chain antibody (HCAb). Subsequently, HCAb was also found in cartilaginous fish such as nurse sharks, rays, white-spotted dogfish, and striped bamboo sharks. Although HCAb lacks a single chain, its function is complete, which indicates that the heavy chain variable region of heavy chain antibodies can form a complete antigen binding site alone.

[0005] Therefore, there is an urgent need to develop a new type of waterproof and pathogenic bacteria product that is not easy to develop drug resistance, has strong specificity, low immunogenicity, and strong stability and permeability. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibodies, preparation methods and applications thereof, which have the characteristics of strong specificity, high affinity and strong stability.

[0007] To solve the above technical problems, the technical solution of the present invention is: anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01, the innovation of which is that the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01 is composed of a framework region, a complementary determining region, and a connecting peptide connecting the framework region and the complementary determining region to form a VHH sequence, and the nucleotide sequence encoding the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01 is shown in SEQ ID NO.9.

[0008] SEQ ID NO: 9

[0009]

[0010] Preferably, the VHH sequence includes, from left to right, an N-terminus at the left end and a C-terminus at the right end;

[0011] The framework region includes, from left to right, an amino acid sequence framework region FR1, an amino acid sequence framework region FR2, an amino acid sequence framework region FR3 and an amino acid sequence framework region FR4, wherein the amino acid sequence framework region FR1 is located at the N-terminus, and the amino acid sequence framework region FR4 is located at the C-terminus;

[0012] The complementary determining regions include, from left to right, an amino acid sequence complementary determining region CDR 1, an amino acid sequence complementary determining region CDR 2 and an amino acid sequence complementary determining region CDR 3, wherein the amino acid sequence complementary determining region CDR 1 is located between the amino acid sequence framework region FR1 and the amino acid sequence framework region FR2, the amino acid sequence complementary determining region CDR 2 is located between the amino acid sequence framework region FR2 and the amino acid sequence framework region FR3, and the amino acid sequence complementary determining region CDR3 is located between the amino acid sequence framework region FR3 and the amino acid sequence framework region FR4;

[0013] The amino acid sequence framework region FR1, the amino acid sequence complementarity determining region CDR 1, the amino acid sequence framework region FR2, the amino acid sequence complementarity determining region CDR 2, the amino acid sequence framework region FR3, the amino acid sequence complementarity determining region CDR 3 and the amino acid sequence framework region FR4, each of which is provided with a connecting peptide between any two adjacent ones, and the connecting peptide connects the amino acid sequences of different regions.

[0014] The amino acid sequence of the framework region FR1 is shown in SEQ ID NO: 1; SEQ ID NO: 1 is MAE VQ LQASGGGFVQPGGSLRLSCAA SG.

[0015] The amino acid sequence of the amino acid sequence framework region FR2 is shown in SEQ ID NO: 2; SEQ ID NO: 2 is M GW FRQAPGKEREFVS AIS.

[0016] The amino acid sequence of the amino acid sequence framework region FR3 is shown in SEQ ID NO: 3; SEQ ID NO: 3 is Y YA DSVKGRFTISRDNSKNTVYLQ MNSLRAEDTATYYCA.

[0017] The amino acid sequence of the amino acid sequence framework region FR4 is shown in SEQ ID NO: 4; SEQ ID NO: 4 is Y WG QGTQVTVS.

[0018] The amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO: 5; SEQ ID NO: 5 is YSSRLDE

[0019] The amino acid sequence of the amino acid sequence complementarity determining region CDR2 is shown in SEQ ID NO: 6; SEQ ID NO: 6 is RFTRYV.

[0020] The amino acid sequence of the complementary determining region CDR 3 is shown in SEQ ID NO: 7; SEQ ID NO: 7 is YFGKEHALYQVQRKW.

[0021] Preferably, the amino acid sequence of the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody VHH-KH-01 is shown in SEQ ID NO: 8, SEQ ID NO: 8 is

[0022]

[0023] The method for preparing the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01 is used to prepare the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01, comprising the following steps:

[0024] S1: Obtaining flagellar sequences of Vibrio alginolyticus and Vibrio parahaemolyticus

[0025] The main structural proteins of flagella of Vibrio parahaemolyticus and Vibrio alginolyticus were expressed and purified by prokaryotic expression system, and then used as anti-query NCBI database to perform B1ast analysis on Vibrio parahaemolyticus and Vibrio alginolyticus, and the common flagellar antigen gene protein sequences of Vibrio parahaemolyticus and Vibrio alginolyticus were obtained;

[0026] S2: Specific primers

[0027] According to the restriction site of qET-22b plasmid and the amino acid sequence of antigen protein, the fragment to be inserted into the plasmid was synthesized by PCR product, and the His tag at the C-terminus of the fusion vector was fused; the pET-22b(+) plasmid vector was selected, the selected plasmid was double-digested by BamHI and HindIII, and then the DNA was purified and recovered, and the target gene PCR product was cloned and connected by T5 exonuclease DNA assembly method, and the 20μL reaction solution after connection was added to 100μL competent cells DH5α, cultured and centrifuged to remove the supernatant, and the residue was evenly spread on LB(Amp+) plate and cultured at 37°C; a plurality of single colonies were picked from the plate after culture in LB(Amp+) liquid culture medium, and 1μL was taken as a bacterial liquid template for PCR amplification;

[0028] The plasmids of the positive clones were extracted from the bacterial suspension using a plasmid mini-extraction kit and sent for sequencing. The recombinant vectors with correct sequencing were saved for future use.

[0029] S3: Expression and purification of flagellar antigen proteins of Vibrio alginolyticus and Vibrio parahaemolyticus

[0030] After adding the identified positive recombinant plasmid to the competent cells for activation, remove part of the supernatant by centrifugation, and spread the remainder on LB (Amp+) plates for culture. Select a single colony and inoculate it into the autoinduction medium. After culture and centrifugation, collect the bacteria, break and lyse them by ultrasonication, and then perform SDS-PAGE analysis on the induced expression of the recombinant protein.

[0031] According to the induction conditions of the above study, the antigen recombinant protein was prepared by expanding the culture. After centrifugation of the bacteria, ultrasonic lysis was obtained to obtain the inclusion body precipitate to be purified. After the inclusion body was resuspended, washed and dissolved, the protein was purified by AKTA purification instrument, and the purified protein was analyzed by SDS-PAGE. TM The concentration of purified antigen protein was determined by BCA protein quantification kit;

[0032] The eluted protein was added to a 10 kDa dialysis bag and dialyzed in the following order: 4 M 2 h, 3 M 2 h, 2 M overnight, 1 M 2 h, 0 M 2 h to remove urea and high concentration imidazole;

[0033] S4: Enrichment and selection of nanoantibodies using phage display technology

[0034] After TG1 activation, a single TG1 colony was inoculated into 5 mL of 2×YT liquid medium and cultured overnight;

[0035] The purified recombinant protein was dissolved in 1× PBS solution and coated and incubated at 37°C for 2 h and 4°C overnight.

[0036] The next day, the cells were washed three times and 5% skim milk was added to block for 1 hour. 1 mL of the laboratory shark-derived nanoantibody synthesis library was taken and 1 mL of 5% skim milk was added to rotate and mix for 1 hour. The mixture was added to the control immune tube and incubated at room temperature for 1 hour. After the phage display library was added to the negative control tube for 1 hour, the experimental group poured out the blocking solution, washed three times, and the phage milk mixture in the control tube was added to incubate for 1 hour. The control group was washed three times and placed at room temperature.

[0037] The liquid of the experimental group and the control group was discarded, and the cells were washed seven times. 0.1 M HCl was added to elute the phages, and 1 M Tris-HCl pH 7.4 was used for neutralization.

[0038] Activate the TG1 bacterial solution. When the OD600 is 0.5, add the eluted phage solutions of the experimental group and the control group to the activated TG1 bacterial solution respectively.

[0039] 10 mL of TG1 bacterial solution was cultured for 1 h;

[0040] For the experimental group and the control group, 10 μL of bacterial solution was taken into a centrifuge tube, diluted with 2×YT medium, and then 100 μL and 10 μL were spread on two 9 cm diameter 2×YT (Amp) solid plates, respectively, and the colony count analysis was performed every other day;

[0041] The remaining experimental bacteria were centrifuged and plated to collect the selected phages;

[0042] The solid plate was washed with 2×YT medium, diluted to determine OD600, and the bacterial solution was added to 2×YT medium according to OD, and Amp antibiotic 100 μg / mL was added for culture, and helper phage M13K07 was added for culture for 1 hour; then Kana antibiotic 50 μg / mL was added and cultured overnight; the next day, the supernatant phage was collected by centrifugation at room temperature, PEG 6000 solution was added, incubated, and the supernatant was removed by centrifugation to obtain phage precipitation;

[0043] Resuspend and dissolve with PBS solution, centrifuge again to remove residual bacteria, take the supernatant and read the 0D268 value, 10D means 5×10 12 pfu / mL;

[0044] Take 0.20D of supernatant for the next round of phage library screening, and freeze the rest at -80°C;

[0045] Repeat 2-4 rounds of the above phage panning and preparation process until the colony counts of the experimental group and the control group on the counting plate are significantly different, and clone identification is performed using phage ELISA;

[0046] S5: Identification of the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody VHH-KH-01

[0047] The plates were coated with recombinant flagellar proteins of Vibrio parahaemolyticus and Vibrio alginolyticus at preset concentrations overnight at 4°C, blocked with 5% skim milk for 2 h, added with 100 μL of phage-displayed nanoantibodies, incubated at room temperature for 1 h, washed 9 times with 1× PBST solution, added with 100 μL of M13 secondary antibody working solution, and incubated for 1 h;

[0048] Wash the plate 9 times, add TMB substrate for color development, incubate for 5-10 min, add 50 μL 0.1 M sulfuric acid to terminate the reaction, and read the OD value of each well at OD450;

[0049] Calculate the P / N value, and the wells with P / N ≥ 2.1 are regarded as positive wells. Extract plasmids from the positive wells and perform sequencing analysis to determine the framework region and complementary region of the antibody sequence;

[0050] S6: Construction of Pichia pastoris nanobody expression system

[0051] The plasmid of phage-displayed nanoantibodies was extracted, linearized with a single-cutting enzyme, and then transformed into Pichia pastoris SMD1168 using LiCl; a single clone was picked the next day, and yeast genomic DNA was extracted with lysis buffer, and the transformation results were detected by electrophoresis after PCR; the positive strains were expanded and cultured overnight using BMGY medium, resuspended in BMMY by centrifugation, and induced by adding methanol with a final concentration of 0.5%; the supernatant and precipitate were collected by centrifugation, and the supernatant was analyzed by SDS-PAGE electrophoresis.

[0052] Preferably, the PCR amplification of the bacterial liquid template in step S2 includes: setting the reaction system and setting the reaction temperature;

[0053] The reaction system was set up to include 1 μL bacterial template, 10 μL 2× Taq Master mix, 1 μL T7, 1 μL T7Ter, and 7 μL deionized water;

[0054] The reaction temperature settings included: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, and 72°C total extension for 10 min.

[0055] A recombinant vector contains a nucleotide sequence encoding the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01 in the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody.

[0056] A host cell, wherein the host cell contains the recombinant vector.

[0057] The beneficial effects of the present invention are as follows: the present invention discloses a shark-derived nano-antibody that targets both Vibrio parahaemolyticus and Vibrio alginolyticus, can specifically identify Vibrio parahaemolyticus and Vibrio alginolyticus, and is not highly specific to other Vibrio. Shark fish have a close evolutionary relationship with aquaculture animals, and the developed nano-antibody has low immunogenicity.

[0058] Immunoglobulin IgG is differentiated by B cells after being stimulated by antigens, and specifically recognizes and binds to antigens. Shark-derived nanobodies are the smallest and most complete antigen-binding fragments discovered so far, with a molecular weight of only 12kDa. Shark-derived nanobodies have the advantages of small molecular weight, high affinity, strong stability, good solubility, strong tissue penetration, etc., and can be recombinantly expressed, which is conducive to mass production and low cost.

[0059] The shark-derived nanoantibody VHH-KH-01 for Vibrio parahaemolyticus and Vibrio alginolyticus provided by the present invention has the advantages of small molecular weight, high affinity, strong stability, good solubility, strong tissue penetration, etc., and can be recombinantly expressed, which is conducive to mass production and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0061] Figure 1 It is a schematic diagram of double enzyme digestion of qET-22b(+) vector in the present invention against Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01, preparation method and application.

[0062] Figure 2 It is a schematic diagram of the positive clone of the recombinant expression vector in the present invention's anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01, its preparation method and application.

[0063] Figure 3 It is a schematic diagram of the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01, its preparation method and VHH induction expression in its application.

[0064] Figure 4 It is a schematic diagram of the agglutination activity of the nanoantibody against Vibrio parahaemolyticus and Vibrio alginolyticus in the present invention, the preparation method and application thereof.

[0065] Figure 5 It is a schematic diagram of the VHH sequence in the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01, the preparation method and the application thereof. DETAILED DESCRIPTION

[0066] The anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01 of the present invention is composed of a framework region, a complementary determining region, and a connecting peptide connecting the framework region and the complementary determining region to form a VHH sequence. The nucleotide sequence encoding the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01 is shown in SEQ ID NO: 9.

[0067] SEQ ID NO: 9 is

[0068]

[0069] The above VHH sequence includes, from left to right, the N-terminus at the left end and the C-terminus at the right end;

[0070] The framework region includes, from left to right, an amino acid sequence framework region FR1, an amino acid sequence framework region FR2, an amino acid sequence framework region FR3 and an amino acid sequence framework region FR4, wherein the amino acid sequence framework region FR1 is located at the N-terminus, and the amino acid sequence framework region FR4 is located at the C-terminus;

[0071] The complementary determining regions include, from left to right, an amino acid sequence complementary determining region CDR 1, an amino acid sequence complementary determining region CDR 2 and an amino acid sequence complementary determining region CDR 3, wherein the amino acid sequence complementary determining region CDR 1 is located between the amino acid sequence framework region FR1 and the amino acid sequence framework region FR2, the amino acid sequence complementary determining region CDR 2 is located between the amino acid sequence framework region FR2 and the amino acid sequence framework region FR3, and the amino acid sequence complementary determining region CDR3 is located between the amino acid sequence framework region FR3 and the amino acid sequence framework region FR4;

[0072] The above-mentioned amino acid sequence framework region FR1, amino acid sequence complementarity determining region CDR 1, amino acid sequence framework region FR2, amino acid sequence complementarity determining region CDR 2, amino acid sequence framework region FR3, amino acid sequence complementarity determining region CDR 3 and amino acid sequence framework region FR4, each of which is provided with a connecting peptide between any two adjacent ones, and the connecting peptide connects the amino acid sequences of different regions.

[0073] The amino acid sequence of the framework region FR1 is shown in SEQ ID NO: 1; SEQ ID NO: 1 is MAE VQ LQASGGGFVQPGGSLRLSCAA SG.

[0074] The amino acid sequence of the amino acid sequence framework region FR2 is shown in SEQ ID NO: 2; SEQ ID NO: 2 is M GW FRQAPGKEREFVS AIS.

[0075] The amino acid sequence of the amino acid sequence framework region FR3 is shown in SEQ ID NO: 3; SEQ ID NO: 3 is Y YA DSVKGRF TIS RDNSKNTVYLQMNSLRAEDTA TYYCA.

[0076] The amino acid sequence of the amino acid sequence framework region FR4 is shown in SEQ ID NO: 4; SEQ ID NO: 4 is Y WG QGTQVTVS.

[0077] The amino acid sequence of the amino acid sequence complementarity determining region CDR 1 is shown in SEQ ID NO: 5; SEQ ID NO: 5 is YSSRLDE.

[0078] The amino acid sequence of the amino acid sequence complementarity determining region CDR2 is shown in SEQ ID NO: 6; SEQ ID NO: 6 is RFTRYV.

[0079] The amino acid sequence of the complementary determining region CDR 3 is shown in SEQ ID NO: 7; SEQ ID NO: 7 is YFGKEHALYQVQRKW.

[0080] The amino acid sequence of the above-mentioned anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody is shown in SEQ ID NO: 8. SEQ ID NO: 8 is

[0081]

[0082] The preparation method of the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody VHH-KH-01 of the present invention comprises the following steps:

[0083] S1: Obtaining flagellar sequences of Vibrio alginolyticus and Vibrio parahaemolyticus

[0084] The main structural proteins of flagella of Vibrio parahaemolyticus and Vibrio alginolyticus were expressed and purified by prokaryotic expression system, and then used as anti-query NCBI database to perform Blast analysis on Vibrio parahaemolyticus and Vibrio alginolyticus, and the common flagellar antigen gene protein sequences of Vibrio parahaemolyticus and Vibrio alginolyticus were obtained;

[0085] S2: Specific primers

[0086] According to the restriction site of qET-22b plasmid and the amino acid sequence of antigen protein, the fragment to be inserted into the plasmid was synthesized by PCR product, and the His tag at the C-terminus of the fusion vector was fused; the pET-22b(+) plasmid vector was selected, the selected plasmid was double-digested by BamHI and HindIII, and then the DNA was purified and recovered, and the target gene PCR product was cloned and connected by T5 exonuclease DNA assembly method, and the 20 μL reaction solution after connection was added to 100 μL competent cells DH5α, cultured and centrifuged to remove the supernatant, and the residue was evenly spread on LB(Amp+) plate and cultured at 37°C; multiple single colonies were picked from the cultured plate in LB(Amp+) liquid culture medium, and 1 μL was taken as a bacterial liquid template for PCR amplification;

[0087] The plasmids of the positive clones were extracted from the bacterial suspension using a plasmid mini-extraction kit and sent for sequencing. The recombinant vectors with correct sequencing were saved for future use.

[0088] S3: Expression and purification of flagellar antigen proteins of Vibrio alginolyticus and Vibrio parahaemolyticus

[0089] After adding the identified positive recombinant plasmid to the competent cells for activation, remove part of the supernatant by centrifugation, and spread the remainder on LB (Amp+) plates for culture. Select a single colony and inoculate it into the autoinduction medium. After culture and centrifugation, collect the bacteria, break and lyse them by ultrasonication, and then perform SDS-PAGE analysis on the induced expression of the recombinant protein.

[0090] According to the induction conditions of the above study, the antigen recombinant protein was prepared by expanding the culture. After centrifugation of the bacteria, ultrasonic lysis was obtained to obtain the inclusion body precipitate to be purified. After the inclusion body was resuspended, washed and dissolved, the protein was purified by AKTA purification instrument, and the purified protein was analyzed by SDS-PAGE. TM The concentration of purified antigen protein was determined by BCA protein quantification kit;

[0091] The eluted protein was added to a 10 kDa dialysis bag and dialyzed in the following order: 4 M 2 h, 3 M 2 h, 2 M overnight, 1 M 2 h, 0 M 2 h to remove urea and high concentration imidazole;

[0092] S4: Enrichment and selection of nanoantibodies using phage display technology

[0093] After TG1 activation, a single TG1 colony was inoculated into 5 mL of 2×YT liquid medium and cultured overnight;

[0094] The purified recombinant protein was dissolved in 1× PBS solution and coated and incubated at 37°C for 2 h and 4°C overnight.

[0095] The next day, the cells were washed three times and 5% skim milk was added to block for 1 hour. 1 mL of the laboratory shark-derived nanoantibody synthesis library was taken and 1 mL of 5% skim milk was added to rotate and mix for 1 hour. The mixture was added to the control immune tube and incubated at room temperature for 1 hour. After the phage display library was added to the negative control tube for 1 hour, the experimental group poured out the blocking solution, washed three times, and the phage milk mixture in the control tube was added to incubate for 1 hour. The control group was washed three times and placed at room temperature.

[0096] The liquid of the experimental group and the control group was discarded, and the cells were washed seven times, 0.1 M HCl was added to elute the phages, and 1 M Tris-HCl pH 7.4 was used for neutralization;

[0097] Activate the TG1 bacterial solution. When the OD600 is 0.5, add the eluted phage solutions of the experimental group and the control group to the activated TG1 bacterial solution respectively.

[0098] 10 mL of TG1 bacterial solution was cultured for 1 h;

[0099] Take 10 μ L of bacterial solution from the experimental group and the control group into a centrifuge tube, dilute it with 2×YT medium, and then spread 100 μ L and 10 μ L onto two 9 cm diameter 2×YT (Amp) solid plates, respectively, and count the colonies every other day;

[0100] The remaining experimental bacteria were centrifuged and plated to collect the selected phages;

[0101] The solid plate was washed with 2×YT medium, diluted to determine OD600, and the bacterial solution was added to 2×YT medium according to OD, and 100 μg / mL of Amp antibiotic was added for culture, and helper phage M13K07 was added for culture for 1 hour; then 50 μg / mL of Kana antibiotic was added and cultured overnight; the next day, the supernatant phage was collected by centrifugation at room temperature, PEG 6000 solution was added, incubated, and the supernatant was removed by centrifugation to obtain phage precipitation;

[0102] Resuspend and dissolve with PBS solution, centrifuge again to remove residual bacteria, take the supernatant and read the 0D268 value, 10D means 5×10 12 pfu / mL;

[0103] Take 0.2OD of supernatant for the next round of phage library screening, and freeze the rest at -80℃;

[0104] Repeat 2-4 rounds of the above phage panning and preparation process until the colony counts of the experimental group and the control group on the counting plate are significantly different, and clone identification is performed using phage ELISA;

[0105] S5: Identification of the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody VHH-KH-01

[0106] The plates were coated with recombinant flagellar proteins of Vibrio parahaemolyticus and Vibrio alginolyticus at preset concentrations overnight at 4°C, blocked with 5% skim milk for 2 h, added with 100 μL of phage-displayed nanoantibodies, incubated at room temperature for 1 h, washed 9 times with 1× PBST solution, added with 100 μL of M13 secondary antibody working solution, and incubated for 1 h;

[0107] Wash the plate 9 times, add TMB substrate for color development, incubate for 5-10 min, add 50 μL 0.1 M sulfuric acid to terminate the reaction, and read the OD value of each well at OD450;

[0108] Calculate the P / N value, and the wells with P / N ≥ 2.1 are regarded as positive wells. Extract plasmids from the positive wells and perform sequencing analysis to determine the framework region and complementary region of the antibody sequence;

[0109] S6: Construction of Pichia pastoris nanobody expression system

[0110] The plasmid of phage-displayed nanoantibodies was extracted, linearized with a single-cutting enzyme, and then transformed into Pichia pastoris SMD1168 using LiCl; a single clone was picked the next day, and yeast genomic DNA was extracted with lysis buffer, and the transformation results were detected by electrophoresis after PCR; the positive strains were expanded and cultured overnight using BMGY medium, resuspended in BMMY by centrifugation, and induced by adding methanol with a final concentration of 0.5%; the supernatant and precipitate were collected by centrifugation, and the supernatant was analyzed by SDS-PAGE electrophoresis.

[0111] The PCR amplification of the bacterial liquid template in the above step S2 includes: setting the reaction system and setting the reaction temperature;

[0112] The reaction system was set up to include 1 μL bacterial template, 10 μL 2× Taq Master mix, 1 μL T7, 1 μL T7Ter, and 7 μL deionized water;

[0113] The reaction temperature settings included: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, and 72°C total extension for 10 min.

[0114] A recombinant vector contains a nucleotide sequence encoding the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01 in the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody.

[0115] A host cell, wherein the host cell contains the recombinant vector.

[0116] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0117] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobodies, characterized in that: The anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody is composed of a framework region, a complementary determining region, and a connecting peptide connecting the framework region and the complementary determining region to form a VHH sequence. The nucleotide sequence encoding the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody VHH-KH-01 is shown in SEQ ID NO.

9.

2. The anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody according to claim 1, characterized in that: The VHH sequence includes, from left to right, an N-terminus at the left end and a C-terminus at the right end; The framework region includes, from left to right, an amino acid sequence framework region FR1, an amino acid sequence framework region FR2, an amino acid sequence framework region FR3 and an amino acid sequence framework region FR4, wherein the amino acid sequence framework region FR1 is located at the N-terminus, and the amino acid sequence framework region FR4 is located at the C-terminus; The complementary determining regions include, from left to right, an amino acid sequence complementary determining region CDR 1, an amino acid sequence complementary determining region CDR 2 and an amino acid sequence complementary determining region CDR 3, wherein the amino acid sequence complementary determining region CDR 1 is located between the amino acid sequence framework region FR1 and the amino acid sequence framework region FR2, the amino acid sequence complementary determining region CDR 2 is located between the amino acid sequence framework region FR2 and the amino acid sequence framework region FR3, and the amino acid sequence complementary determining region CDR3 is located between the amino acid sequence framework region FR3 and the amino acid sequence framework region FR4; The amino acid sequence framework region FR1, the amino acid sequence complementarity determining region CDR 1, the amino acid sequence framework region FR2, the amino acid sequence complementarity determining region CDR 2, the amino acid sequence framework region FR3, the amino acid sequence complementarity determining region CDR 3 and the amino acid sequence framework region FR4, each of which is provided with a connecting peptide between any two adjacent ones, and the connecting peptide connects the amino acid sequences of different regions.

3. The anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody according to claim 2, characterized in that: The amino acid sequence of the amino acid sequence framework region FR1 is shown in SEQ ID NO: 1; The amino acid sequence of the amino acid sequence framework region FR2 is shown in SEQ ID NO: 2; The amino acid sequence of the amino acid sequence framework region FR3 is shown in SEQ ID NO: 3; The amino acid sequence of the amino acid sequence framework region FR4 is shown in SEQ ID NO: 4; The amino acid sequence of the complementary determining region CDR 1 is shown in SEQ ID NO: 5; The amino acid sequence of the complementary determining region CDR 2 is shown in SEQ ID NO: 6; The amino acid sequence of the amino acid sequence complementarity determining region CDR 3 is shown in SEQ ID NO:

7.

4. The anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody according to claim 1, characterized in that: The amino acid sequence of the anti-Vibrio parahaemolyticus and anti-Vibrio alginolyticus nanobody VHH-KH-01 is shown in SEQ ID NO:

8.

5. A method for preparing an anti-Vibrio parahaemolyticus and anti-Vibrio alginolyticus nanobody, which is used to prepare an anti-Vibrio parahaemolyticus and anti-Vibrio alginolyticus nanobody as claimed in any one of claims 1 to 4, characterized in that The following steps are involved: S1: Obtaining flagellar sequences of Vibrio alginolyticus and Vibrio parahaemolyticus The main structural proteins of flagella of Vibrio parahaemolyticus and Vibrio alginolyticus were expressed and purified by prokaryotic expression system, and then used as anti-query NCBI database to perform Blast analysis on Vibrio parahaemolyticus and Vibrio alginolyticus, and the common flagellar antigen gene protein sequences of Vibrio parahaemolyticus and Vibrio alginolyticus were obtained; S2: Specific primers According to the restriction site of qET-22b plasmid and the amino acid sequence of antigen protein, the fragment to be inserted into the plasmid was synthesized by PCR product, and the His tag at the C-terminus of the fusion vector was fused; the pET-22b(+) plasmid vector was selected, the selected plasmid was double-digested by BamHI and HindIII, and then the DNA was purified and recovered, and the target gene PCR product was cloned and connected by T5 exonuclease DNA assembly method, and the 20μL reaction solution after connection was added to 100μL competent cells DH5α, cultured and centrifuged to remove the supernatant, and the residue was evenly spread on LB(Amp+) plates and cultured at 37°C; a plurality of single colonies were picked from the cultured plates and placed in LB(Amp+) liquid culture medium, and 1μL was taken as a bacterial liquid template for PCR amplification; The plasmids of the positive clones were extracted from the bacterial suspension using a plasmid mini-extraction kit and sent for sequencing. The recombinant vectors with correct sequencing were saved for future use. S3: Expression and purification of flagellar antigen proteins of Vibrio alginolyticus and Vibrio parahaemolyticus After adding the identified positive recombinant plasmid to the competent cells for activation, remove part of the supernatant by centrifugation, and spread the remainder on LB (Amp+) plates for culture. Select a single colony and inoculate it into the autoinduction medium. After culture and centrifugation, collect the bacteria, break and lyse them by ultrasonication, and then perform SDS-PAGE analysis on the induced expression of the recombinant protein. According to the induction conditions of the above study, the antigen recombinant protein was prepared by expanding the culture. After centrifugation of the bacteria, ultrasonic lysis was obtained to obtain the inclusion body precipitate to be purified. After the inclusion body was resuspended, washed and dissolved, the protein was purified by AKTA purification instrument, and the purified protein was analyzed by SDS-PAGE. TM The concentration of purified antigen protein was determined by BCA protein quantification kit; The eluted protein was added to a 10 kDa dialysis bag and dialyzed in the following order: 4 M 2 h, 3 M 2 h, 2 M overnight, 1 M 2 h, 0 M 2 h to remove urea and high concentration imidazole; S4: Enrichment and selection of nanoantibodies using phage display technology After TG1 activation, a single TG1 colony was inoculated into 5 mL of 2×YT liquid medium and cultured overnight; The purified recombinant protein was dissolved in 1× PBS solution and coated and incubated at 37°C for 2 h and 4°C overnight. The next day, wash three times, add 5% skim milk, block for 1 hour, take 1mL of laboratory shark-derived nanoantibody synthesis library, add 1mL of 5% skim milk, rotate and mix for 1 hour, then add the mixture to the control immune tube and incubate at room temperature for 1 hour; add the phage display library to the negative control tube for 1 hour, pour out the blocking solution in the experimental group, wash three times, add the phage milk mixture in the control tube, incubate for 1 hour, and wash the control group three times and leave at room temperature; The liquid of the experimental group and the control group was discarded, and the cells were washed seven times, 0.1 M HCl was added to elute the phages, and 1 M Tris-HCl pH 7.4 was used for neutralization; Activate the TG1 bacterial solution. When the OD600 is 0.5, add the eluted phage solutions of the experimental group and the control group to the activated TG1 bacterial solution respectively. 10 mL of TG1 bacterial solution was cultured for 1 h; Take 10 μL of bacterial solution from the experimental group and the control group into a centrifuge tube, dilute it with 2×YT medium, and then spread 100 μL and 10 μL onto two 9 cm diameter 2×YT (Amp) solid plates, respectively, and count the colonies every other day; The remaining experimental bacteria were centrifuged and plated to collect the selected phages; The solid plate was washed with 2×YT medium, diluted to determine OD600, and the bacterial solution was added to 2×YT medium according to OD, and Amp antibiotic 100 μg / mL was added for culture, and helper phage M13K07 was added for culture for 1 hour; then Kana antibiotic 50 μg / mL was added and cultured overnight; the next day, the supernatant phage was collected by centrifugation at room temperature, PEG 6000 solution was added, incubated, and the supernatant was removed by centrifugation to obtain phage precipitation; Resuspend and dissolve with PBS solution, centrifuge again to remove residual bacteria, take the supernatant and read the 0D268 value, 10D means 5×10 12 pfu / mL; Take 0.20D of supernatant for the next round of phage library screening, and freeze the rest at -80°C; Repeat 2-4 rounds of the above phage panning and preparation process until the colony counts of the experimental group and the control group on the counting plate are significantly different, and clone identification is performed using phage ELISA; S5: Identification of the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody VHH-KH-01 The plates were coated with recombinant flagellar proteins of Vibrio parahaemolyticus and Vibrio alginolyticus at preset concentrations overnight at 4°C, blocked with 5% skim milk for 2 h, added with 100 μL of phage-displayed nanoantibodies, incubated at room temperature for 1 h, washed 9 times with 1× PBST solution, added with 100 μL of M13 secondary antibody working solution, and incubated for 1 h; Wash the plate 9 times, add TMB substrate for color development, incubate for 5-10 min, add 50 μL 0.1 M sulfuric acid to terminate the reaction, and read the OD value of each well at OD450; Calculate the P / N value, and the wells with P / N ≥ 2.1 are regarded as positive wells. Extract plasmids from the positive wells and perform sequencing analysis to determine the framework region and complementary region of the antibody sequence; S6: Construction of Pichia pastoris nanobody expression system The plasmid of phage-displayed nanoantibodies was extracted, linearized with a single cutter, and then transformed into Pichia pastoris SMD1 168 using LiCl. A single clone was picked the next day, and yeast genomic DNA was extracted with lysis buffer. The transformation results were detected by electrophoresis after PCR. The positive strains were expanded and cultured overnight using BMGY medium, resuspended in BMMY by centrifugation, and induced by adding methanol with a final concentration of 0.5%. The supernatant and precipitate were collected by centrifugation, and the supernatant was analyzed by SDS-PAGE electrophoresis.

6. The method for preparing the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanobody according to claim 5, characterized in that: The PCR amplification of the bacterial liquid template in step S2 includes: setting the reaction system and setting the reaction temperature; The reaction system was set up to include 1 μL bacterial template, 10 μL 2× Taq Master mix, 1 μL T7, 1 μL T7Ter, and 7 μL deionized water; The reaction temperature settings included: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, and 72°C total extension for 10 min.

7. A recombinant vector, characterized in that: The recombinant vector contains the nucleotide sequence encoding the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody VHH-KH-01 in the anti-Vibrio parahaemolyticus and Vibrio alginolyticus nanoantibody according to claim 1.

8. A host cell, characterized in that The host cell contains the recombinant vector according to claim 7.

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