CN113461814B

CN113461814B - Nano antibody for specifically recognizing vibrio parahaemolyticus, recombinant vector, host cell and application thereof

Info

Publication number: CN113461814B
Application number: CN202110721410.5A
Authority: CN
Inventors: 王妍入; 王鹏; 廖星蕊; 张瑶; 魏娟; 王建龙; 马敏
Original assignee: Northwest A&F University
Current assignee: Northwest A&F University
Priority date: 2021-06-28
Filing date: 2021-06-28
Publication date: 2022-05-27
Anticipated expiration: 2041-06-28
Also published as: CN113461814A

Abstract

The invention provides a nano antibody for specifically recognizing vibrio parahaemolyticus, which comprises a coding gene of the nano antibody, and also provides a vector and a host cell containing a nucleotide sequence for coding the nano antibody, wherein the amino acid sequence of the nano antibody is shown as SEQ ID NO:1, and the nano antibody has good specificity and binding capacity for vibrio parahaemolyticus. The nano antibody for specifically identifying the vibrio parahaemolyticus provided by the invention has the advantages of good specificity, strong stability and high expression yield, is not combined with other non-vibrio parahaemolyticus, and can specifically identify the vibrio parahaemolyticus.

Description

Nano antibody for specifically recognizing vibrio parahaemolyticus, recombinant vector, host cell and application thereof

Technical Field

The invention belongs to the field of microorganism detection. Specifically, the invention relates to a nano antibody, a recombinant vector, a host cell and application thereof for specifically recognizing vibrio parahaemolyticus.

Background

Vibrio parahaemolyticus is a food-borne pathogen and can cause abdominal pain, vomiting and diarrhea clinically. Eating food containing the bacteria can cause food poisoning, also called halophilic bacteria food poisoning. The vibrio parahaemolyticus is gram-negative bacillus, is in various shapes such as arc, rod and filiform, and has no odontoblast. It is also a marine bacterium, and is mainly derived from marine products such as fish, shrimp, crab, shellfish and seaweed. With the increasing market demand of marine products, a rapid and sensitive detection method is very important for ensuring the safety of food supply in order to ensure the safety of food processing links from fresh food to dining tables and low infection of marine products.

At present, many detection methods are researched aiming at vibrio parahaemolyticus, and the traditional methods comprise a plate culture method, a cell counting method, a bioluminescence method, an impedance measurement method and an immunological method. The most widely used at present are plate counting methods and immunological methods based on antigen-antibody reactions. Based on the growth principle of microorganisms, the traditional food pathogen detection method must be carried out in a microorganism laboratory, and usually requires complex sample treatment, the process is complicated, and the result can be obtained in 4 to 7 days generally. The immunological method refers to a detection method based on an antigen-antibody specific binding reaction. In order to detect specific microorganisms and toxins thereof, antibodies are widely used in various immunological detection methods. In the method, most antibodies are derived from mouse or rabbit serum, the main evaluation indexes of the method comprise specificity and sensitivity, and the types of the antibodies comprise polyclonal antibodies and monoclonal antibodies. Although the preparation process of the polyclonal antibody is simple in operation, the preparation period is short, and the cost is low. However, the use of polyclonal antisera also has its own drawbacks, as polyclonal antibodies are less specific due to variability and contingency of the animal immune response. Monoclonal antibodies require a large amount of cost and time, and the titer of antibodies in different batches varies, and the production of these antibodies does not meet the current trend of animal welfare, which has led to an increasing interest in genetically engineered antibodies.

The phage display nanometer antibody technology is characterized in that an exogenous gene segment is inserted into a gene segment of a phage and fusion expression is carried out on a capsid protein shell of the phage, and the method is simple and rapid and can be used for mass production. While nanobody, an antibody lacking a heavy chain and comprising only a light chain, has a molecular weight that is one tenth of that of conventional antibodies, and is the smallest unit known to bind to an antigen, the longer CDR3 region of nanobody makes it possible to recognize the cryptic region of the antigen. The present company has filed for patent inventions: CN 112707963A-salmonella broad-spectrum recognition nano antibody, recombinant vector, host cell and application thereof, and the provided anti-salmonella nano antibody has good broad-spectrum property, strong stability and high expression yield.

At present, no nano antibody aiming at vibrio parahaemolyticus exists in the existing nano antibodies, and a detection technology for the vibrio parahaemolyticus by applying the nano antibody is provided.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a nano antibody with good specificity, recognition capability and binding capability to vibrio parahaemolyticus, and the nano antibody is used as a capture antibody and a detection antibody and applied to the detection technology of vibrio parahaemolyticus, and is realized by the following technology.

The nano antibody specifically recognizing the vibrio parahaemolyticus has an amino acid sequence comprising amino acid sequence framework regions FR1, FR2, FR3 and FR4, and amino acid sequence complementarity determining regions CDR1, CDR2 and CDR 3;

the amino acid sequence of the amino acid sequence framework region FR1 (the first constant region sequence of the nanobody) is shown as SEQ ID NO. 3; the amino acid sequence of the amino acid sequence framework region FR2 (second constant region sequence of the nano antibody) is shown as SEQ ID NO. 4; the amino acid sequence of the amino acid sequence framework region FR3 (the third constant region sequence of the nanobody) is shown as SEQ ID NO. 5; the amino acid sequence of the amino acid sequence framework region FR4 (the fourth segment constant region sequence of the nanobody) is shown as SEQ ID NO. 6;

the amino acid sequence of the amino acid sequence complementarity determining region CDR1 (the first variable region sequence of the nanobody) is shown as SEQ ID NO. 7; the amino acid sequence of the amino acid sequence complementarity determining region CDR2 (the second variable region sequence of the nanobody) is shown as SEQ ID NO. 8; the amino acid sequence of the amino acid sequence complementarity determining region CDR3 (the third variable region sequence of nanobody) is shown in SEQ ID NO. 9.

The nano antibody is an antibody with good binding capacity and specificity to vibrio parahaemolyticus. At present, monoclonal antibodies and polyclonal antibodies aiming at vibrio parahaemolyticus have poor stability, long preparation period and high preparation cost, and are easy to combine with other bacteria.

The enzyme-linked immunoassay method of the double-nano antibody sandwich established by the nano antibody can specifically detect vibrio parahaemolyticus, and has a better detection result. The invention can solve the problems of poor specificity and higher cost of the existing detection method, and enables the enzyme-linked immunoassay method of the double-nano antibody sandwich to be more widely applied.

Preferably, the amino acid sequence of the nano antibody for specifically recognizing the vibrio parahaemolyticus is shown in SEQ ID NO. 1.

Preferably, the nucleotide sequence for coding the nano antibody is shown as SEQ ID NO. 2. The nano antibody with the corresponding amino acid sequence can be synthesized by transcription and translation by utilizing the nucleotide sequence.

The invention also provides an application method of the nano antibody, namely a detection kit for specifically identifying the vibrio parahaemolyticus, which contains the nano antibody for specifically identifying the vibrio parahaemolyticus.

For example, the double-nano antibody sandwich enzyme-linked immunoassay detection kit is prepared by using the nano antibody as a detection reagent, the nano antibody for specifically recognizing vibrio parahaemolyticus is selected as a capture antibody, and the nano antibody displayed by a phage is used as a detection antibody to detect the vibrio parahaemolyticus by using the double-antibody sandwich enzyme-linked immunoassay method. The detection limit of the vibrio parahaemolyticus by adopting the kit is 4.29 multiplied by 10⁶CFU/mL。

The invention also provides a recombinant vector which contains the nucleotide sequence of the coding nano antibody.

A host cell containing the above recombinant vector.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention discloses a special anti-vibrio parahaemolyticus nano antibody, which is not combined with other non-vibrio parahaemolyticus, can specifically identify vibrio parahaemolyticus, can be combined with different sites of vibrio parahaemolyticus, and has the advantages of strong combining ability, good specificity and high thermal stability, and the nano antibody can still keep better ability of combining with antigen under the condition of 37-80 ℃.

(2) The special vibrio parahaemolyticus resistant nano antibody provided by the invention has the advantages of strong stability, short preparation period, low preparation cost, good specificity and high expression yield, and solves the problems that the existing polyclonal antibody of vibrio parahaemolyticus is poor in specificity, a monoclonal antibody needs large cost and time, the titer of antibodies in different batches is different and the antibodies are easy to combine with other bacteria.

(3) The invention solves the problems of poor specificity and higher cost of the vibrio parahaemolyticus detection method, and the established enzyme-linked immunoassay method of the double-nano antibody sandwich can be applied to the vibrio parahaemolyticus detection in marine products, has accurate detection result, and can be widely applied.

Drawings

FIG. 1 is a diagram showing electrophoretic identification of VHH gene amplified by the first round PCR of the example;

FIG. 2 is a diagram showing electrophoretic identification of VHH gene by the second round PCR amplification of the example;

FIG. 3 shows the results of the positive clone identification ELISA by panning;

FIG. 4 is an SDS-PAGE electrophoresis of the anti-Vibrio parahaemolyticus nano antibody Vp + Nb 1;

FIG. 5 is the thermal stability analysis of the anti-Vibrio parahaemolyticus nanobody Vp + Nb 1;

FIG. 6 is the specificity analysis of the anti-Vibrio parahaemolyticus nanobody Vp + Nb 1;

FIG. 7 is a standard curve of the anti-Vibrio parahaemolyticus nano antibody Vp + Nb1 for detecting Vibrio parahaemolyticus.

Detailed Description

The technical solutions of the present invention will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

Examples

1. Construction of phage display nano antibody library of anti-vibrio parahaemolyticus

1.1 camel immunization:

selecting a healthy camel which is not immunized by any antigen, and adding an emulsified mixture of inactivated vibrio parahaemolyticus and Freund complete adjuvant with the same volume of 10⁸CFU/mL concentration for bactrian camel priming, the subsequent immunization adopts Freund incomplete adjuvant to emulsify antigen, and booster immunization is carried out for 1 time every 2 weeks for 6 times. 1 week after each immunization, camel blood was collected to detect antibody titer in serum.

1.2 extraction of total RNA from blood: after the fifth immunization, camel peripheral blood is taken, and total RNA is extracted according to the operation steps of the RNA extraction kit.

1.3 obtaining cDNA by reverse transcription:

oligo (dT) using the total RNA obtained as a template₁₅For reverse transcription of the primer, the first strand of cDNA was synthesized to obtain a cDNA library.

1.4 amplification of Nanobody (VHH) gene fragments:

first round of PCR amplification was performed using the synthesized cDNA as template and CALL001 and CALL002 as primers.

The reaction system is as follows:

10×Ex taq Buffer，5μL

50mM MgSO4，2μL

10mM dNTP，1μL

10mM CALL001 primer, 1. mu.L

10mM CALL002 primer, 1. mu.L

Ex taq DNA polymerase, 0.1. mu.L

cDNA template, 2. mu.L

ddH₂Supplementing O to the total system, 50 μ L;

vortex mixing, centrifuging for a short time, and performing a first round of PCR amplification reaction under the following PCR conditions:

(1)94℃，2min；

(2)94℃，30s；

(3)55℃，30s；

(4)68℃，1min；

30 cycles of amplification in the PCR steps (2) - (4);

(5)68℃，5min。

in the above scheme, the forward primer CALL001 (shown in SEQ ID NO:10) of the PCR amplification VHH is:

5’-GTCCTGGCTGCTCTTCTACAAGG-3’

the reverse primer CALL002 (see SEQ ID NO:11) was:

CALL002:5’-GGTACGTGCTGTTGAACTGTTCC-3’

after the PCR products are separated by 1% agarose gel electrophoresis, DNA fragments with the size of 700bp are purified and recovered by a kit, and the first round of PCR amplification VHH genes are shown in a specific electrophoresis identification picture shown in figure 1, wherein M in the picture represents DL2000marker, and 1 represents the first round of PCR amplification VHH gene products.

And performing second round PCR amplification by using the VHH gene product amplified by the first round PCR as a template and using the CAM-FOR and the CAM-BACK as primers.

The reaction system is as follows:

10×Ex taq Buffer，5μL

50mM MgSO4，2μL

10mM dNTP，1μL

10mM CAM-FOR primer, 1. mu.L

10mM CAM-BACK primer, 1. mu.L

Ex taq DNA polymerase, 0.1. mu.L

cDNA template, 2. mu.L

ddH₂Supplementing O to the total system, 50 μ L;

vortex mixing, after short-time centrifugation, carrying out a second round of PCR amplification reaction, wherein the PCR conditions are as follows:

(1)94℃，2min；

(2)94℃，30s；

(3)55℃，30s；

(4)68℃，1min；

amplifying for 20 cycles in the PCR steps (2) - (4);

(5)68℃，5min。

in the above scheme, the forward primer CAM-FOR (shown in SEQ ID NO:12) of the PCR amplification VHH is:

CAM-FOR：

5’-CATGCCATGACTGTGGCCCAGGCGGCCCAGGTGCAGCTCGTGGAGTCTGGRGGAGG-3’

the reverse primer CAM-BACK (see SEQ ID NO:13) is:

CAM-BACK:5’-CATGCCATGACTCGCGGCCGGCCTGGCCGGAGACGGTGACCAGGGT-3’

after the PCR product is separated by 1% agarose gel electrophoresis, a DNA fragment with the size of 400bp is purified and recovered by a kit, namely a VHH fragment, and a specific electrophoresis chart is shown in figure 2, wherein M in figure 2 represents DL2000marker, and 1 represents a VHH gene product amplified by the second round of PCR.

1.5 construction of the vector

Digestion treatment of pComb3 xss:

the reaction solution was prepared as follows:

pComb3xss vector，2μL

Sfi I，1μL

10×Buffer，2μL

ddH₂supplementing O to the whole system, 20 μ L

After the enzyme digestion product is separated by 1 percent agarose gel electrophoresis, a vector fragment with the size of 3400bp is purified and recovered by a kit.

The VHH gene is connected with the pComb3xss vector subjected to double enzyme digestion treatment, and In-Fusion connection is carried out according to the following system:

digested pComb3xss vector, 1.4. mu.g

VHH gene, 495ng

10×buffer，20μL

T4 ligase，10μL

ddH₂Supplementing O to the whole system, 200 μ L

The reaction was carried out overnight at 16 ℃ for 16h, recovered with agarose gel DNA purification kit and stored at-20 ℃ until use.

1.6 electrotransformation of ligation products

Melting E.coli ER2738 competent cells on ice, adding 3. mu.L of the ligation product into 50. mu.L of the competent cells, gently mixing the competent cells uniformly, quickly transferring the mixture into a precooled electric transfer cup, and placing the electric transfer cup on a Bio-rad electric transfer instrument for electric transfer, wherein the electric transfer conditions are as follows: 1.8kV, 200. omega., 25. mu.F, 1mL of pre-warmed SOC broth was added to the cuvette immediately after electrotransformation, pipetted and transferred to a clean sterile 1.5mL shake tube. Ten times of electrotransformation were carried out as described above, and the bacteria solutions after ten times of electrotransformation were mixed and thawed by gentle shaking at 37 ℃ for 1 hour.

1.7 construction of phage display Nanobody library against Vibrio parahaemolyticus

Transferring the recovered bacteria liquid into 200mLSB culture medium, shaking at 37 deg.C and 250rpm until OD600 value is 0.5, adding 1mL of 1 × 10¹¹pfu of the helper phage M13KO7, 37 ℃ after 1h standing, add kanamycin to a final concentration of 70. mu.g/mL, and shake overnight. The next day, the overnight bacteria were centrifuged at 10000rpm for 15min at 4 ℃, the supernatant was transferred to a sterile centrifuge bottle, 1/5 volumes of PEG/NaCl was added, after standing on ice for 2h, the supernatant was centrifuged at 12000rpm for 20min at 4 ℃, 10mL of sterile 0.5% and BSA in PBS buffer was used to resuspend the precipitate, and the precipitate was dissolved to obtain the amplified anti-Vibrio parahaemolyticus phage display nanobody library.

2. Elutriation and identification of anti-vibrio parahaemolyticus nano antibody

2.1 elutriation of the anti-vibrio parahaemolyticus nano antibody:

inactivated vibrio parahaemolyticus is used as a coating antigen, each well is 100 mu L, and the concentration is 10⁸CFU/ml (concentration of coated bacteria decreased in rounds), 300. mu.L of 3% skimmed milk powder was added for 1h, the plate was washed 3 times with PBST solution containing 0.05%, Tween-20, 100. mu.L of phage display nanobody library was added to each well, incubated at 37 ℃ for 1h, the plate was washed 6 times, 100. mu.L of glycine solution (pH 2.2) was added for elution, and Tris-HCL was immediately added to neutralize the eluted phage. The titer was determined by taking 10. mu.L of eluted phage, and the remaining strain of ER2738, which was used for infection culture to log phase, was amplified, and the amplified phage was immediately used for subsequent panning. In the second and third elutriation rounds, the concentration of coating antigen is1×10⁸CFU/ml, the remaining panning process was the same as the first panning, for a total of 3 panning rounds. After the third round of panning, 10 μ L of phage was taken to determine titer, and the next day, 50 clones were randomly picked up on the plate for phage amplification, and the amplified phage were identified for positive clones by indirect ELISA.

2.2 identification of the anti-vibrio parahaemolyticus nano antibody:

and carrying out positive clone identification on the panned phage display nano antibody by indirect ELISA. The specific operation is as follows: coating the vibrio parahaemolyticus with certain concentration for one night at 4 ℃, sealing the vibrio parahaemolyticus with 3% skimmed milk powder, adding 100 mu L of phage-displayed nano antibody, standing the mixture for 1h at 37 ℃, discarding the supernatant, washing the plate for 6 times with 0.05% PBST solution, adding 100 mu L of enzyme-labeled anti-M13 secondary antibody, and incubating the plate for 1h at 37 ℃. Washing the plate for 6 times, adding TMB substrate for color development, incubating for 15min, adding 50 μ LH₂SO₄The reaction was stopped and the OD of each well was read at 450nm as shown in FIG. 3. And calculating a P/N value, and taking the hole with the P/N value more than or equal to 2.1 as a positive hole for sequencing analysis.

The concentration of the coating antigen of each round of panning is decreased, nano antibodies with higher affinity can be panned, positive clones capable of being combined with vibrio parahaemolyticus are obtained through indirect ELISA screening, sequencing results are analyzed by Bioedit software, IMGT website (http:// www.imgt.org /) is logged in, antibody gene sequences are analyzed, and the framework region and the complementary determining region of the antibody sequences are determined.

3. Preparation of anti-vibrio parahaemolyticus nano antibody

3.1 preparation of phage-displayed Nanobody phage + Nb28 by means of phage amplification

The specific operation steps are as follows: culturing E.coli ER2738 competent cells (100 mL) at 37 deg.C under shaking at 200rpm until OD600 is about 0.6, adding 10 μ L of elutriated phage-displayed nano antibody phage + Nb28, adding 1mL of helper phage M13KO7 (multiplicity of infection is 20: 1), standing at 37 deg.C for 30min, and culturing at 37 deg.C under 250rpm overnight; the next day, the supernatant was collected by centrifugation, 1/5 volumes of PEG-NaCl solution were added, the mixture was inverted and mixed well, and then phages were precipitated; the pellet was collected by centrifugation to obtain phage-displayed nanobodies, leaving 10 μ L for titer determination.

3.2 preparation of soluble Nanobody Vp + Nb1 in protein expression mode

Plasmids of phage display nanobody Vp + Nb1 were extracted and heat shock transformed into expression strain TOP 10F'. The next day, single colonies on the plates were picked for expansion culture. When OD600 is 0.6, IPTG is added to induce expression, the next day, thallus precipitate is collected by centrifugation, cell lysate is added to crack cells, soluble protein is collected, and the nano antibody is identified by nickel column purification and SDS-PAGE.

The results are shown in FIG. 4, where M in FIG. 4 represents protein marker; the second well in FIG. 4 represents the anti-Vibrio parahaemolyticus nanobody Vp + Nb 1. The concentration of the nano antibody is measured by using the Nanodrop, and the yield of the nano antibody is 10mg/L of the culture medium through calculation.

The amino acid sequence of the amino acid sequence framework region FR1 of the vibrio parahaemolyticus nano antibody Vp + Nb1 is shown as SEQ ID NO. 3;

the amino acid sequence of the amino acid sequence framework region FR2 of the vibrio parahaemolyticus nano antibody Vp + Nb1 is shown as SEQ ID NO. 4;

the amino acid sequence of the amino acid sequence framework region FR3 of the vibrio parahaemolyticus nano antibody Vp + Nb1 is shown as SEQ ID NO. 5;

the amino acid sequence of the amino acid sequence framework region FR4 of the vibrio parahaemolyticus nano antibody Vp + Nb1 is shown as SEQ ID NO. 6;

the amino acid sequence of the complementary determining region CDR1 of the amino acid sequence of the vibrio parahaemolyticus nano antibody Vp + Nb1 is shown as SEQ ID NO. 7;

the amino acid sequence of the complementary determining region CDR2 of the amino acid sequence of the vibrio parahaemolyticus nano antibody Vp + Nb1 is shown as SEQ ID NO. 8;

the amino acid sequence of the complementary determining region CDR3 of the amino acid sequence of the vibrio parahaemolyticus nano antibody Vp + Nb1 is shown as SEQ ID NO. 9;

the amino acid sequence of the vibrio parahaemolyticus nano antibody Vp + Nb1 is shown in SEQ ID NO 1;

the nucleotide sequence of the anti-vibrio parahaemolyticus nano antibody Vp + Nb1 is shown in SEQ ID NO. 2.

4. Analysis of thermal stability of anti-vibrio parahaemolyticus nano-antibody

Coating inactivated vibrio parahaemolyticus with a certain concentration on an enzyme label plate, coating overnight at 4 ℃, washing the plate for 3 times on the next day by PBST, adding a nano antibody solution which is processed for 15min at 37 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃, incubating for 1h at 37 ℃, washing the plate for 3 times by PBST, adding an anti-HA enzyme-labeled secondary antibody, incubating for 1h at 37 ℃, washing the plate for 6 times by PBST, adding a TMB substrate, developing for 15min, and using 2MH for developing₂SO₄The reaction was stopped and the absorbance was measured at 450 nm. The effect of different temperature treatments on the activity of the nano antibody is compared, the result is shown in fig. 5, and the nano antibody can keep better antigen binding ability under the condition of 37-80 ℃, which indicates that the nano antibody has certain thermal stability.

5. Specificity analysis of anti-vibrio parahaemolyticus nano antibody

By using a double-nano-antibody sandwich method, one vibrio parahaemolyticus and 5 non-vibrio parahaemolyticus are used as analytes, and the binding capacity of the nano-antibody Vp + Nb1 and other food-borne pathogenic bacteria is measured. Coating 15 mu g/mL of vibrio parahaemolyticus resistant nano antibody on an enzyme label plate at 4 ℃, adding 300 mu L of skimmed milk powder into each hole on the next day, sealing for 1h, washing the plate for 3 times, adding vibrio parahaemolyticus with a certain concentration, enterobacter sakazakii, shigella flexneri, listeria, salmonella and 5 kinds of non-vibrio parahaemolyticus of escherichia coli, incubating for 1h, washing the plate for 3 times, adding 100 mu L of phage displayed nano antibody, incubating for 1h at 37 ℃, washing the plate for three times by PBST, adding an anti-M13-HRP secondary antibody, standing for 1h at 37 ℃, washing the plate for six times, adding a TMB solution, developing for 15min, adding a sulfuric acid solution, stopping the reaction, and judging the specificity of the nano antibody by measuring the absorbance value of each hole under 450 nm. The result is shown in FIG. 6, and the nano antibody Vp + Nb1 is not combined with other 5 kinds of non-vibrio parahaemolyticus. The experimental result shows that the nano antibody Vp + Nb1 can specifically recognize vibrio parahemolyticus, and has strong binding capacity and good specificity.

6. Enzyme-linked immunoassay method for establishing double-nano antibody sandwich

According to the screening pairing, the nano antibody Vp + Nb1 of the vibrio parahaemolyticus is selected as a capture antibody, and the nano antibody displayed by the phageThe antibody phage + Nb28 is used as a detection antibody to carry out double-antibody sandwich enzyme-linked immunoassay to detect the vibrio parahaemolyticus. The method comprises the following specific steps: coating a capture antibody Vp + Nb1 on a 96-hole enzyme label plate, wherein the coating concentration of each hole is 15 mug/mL, and the temperature is 4 ℃ overnight; the next day, the supernatant was discarded, the plate was washed three times with 0.05% PBST, and then blocked with 3% skimmed milk powder for 1h, and Vibrio parahaemolyticus having a concentration of 7.81X 10 was gradiently diluted⁵～1×10⁸CFU/mL, 100. mu.L of bacterial suspension at different concentrations was added to each well and incubated at 37 ℃ for 1 h. Washing the plate with 0.05% PBST three times, adding 100 mu L phage-displayed nano antibody phage + Nb28, incubating for 1h at 37 ℃, washing the plate three times, adding an enzyme-labeled secondary antibody resisting M13, and incubating for 1h at 37 ℃. Washing the plate for six times, adding TMB substrate color development solution, developing for 15min at room temperature, adding 2M sulfuric acid solution to terminate the reaction, detecting the OD value of each well at 450nm, and drawing a standard curve, wherein the standard curve is shown in figure 7. The detection limit of the method is 4.29 multiplied by 10⁶CFU/mL。

Sequence listing

<110> northwest agriculture and forestry science and technology university

<120> nano antibody for specifically recognizing vibrio parahaemolyticus, recombinant vector, host cell and application thereof

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<170> SIPOSequenceListing 1.0

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Ala Ala Leu Val Thr Gly Gly Thr Ala Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met Tyr Tyr Cys

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Ala Leu Gly Ala Ala Trp Thr Cys Ile Asp Phe Thr Arg Arg Arg Ser

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Ala Asp Phe Ser Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

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ccagggaagg agcgcgaggg ggtcgcagcc cttgtaactg gtggtactgc cacatactat 180

gccgactccg tgaagggccg attcaccatc tcccaagaca acgccaagaa cacgttatat 240

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Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly

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Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

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Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn

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Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Met

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Tyr Tyr Cys Ala

35

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catgccatga ctcgcggccg gcctggccgg agacggtgac cagggt 46

Claims

1. The nano antibody for specifically recognizing the vibrio parahaemolyticus is characterized in that the amino acid sequence of the nano antibody comprises amino acid sequence framework regions FR1, FR2, FR3 and FR4, and amino acid sequence complementarity determining regions CDR1, CDR2 and CDR 3;

the amino acid sequence of the amino acid sequence framework region FR1 is shown as SEQ ID NO. 3; the amino acid sequence of the amino acid sequence framework region FR2 is shown as SEQ ID NO. 4; the amino acid sequence of the amino acid sequence framework region FR3 is shown as SEQ ID NO. 5; the amino acid sequence of the amino acid sequence framework region FR4 is shown as SEQ ID NO. 6;

the amino acid sequence of the amino acid sequence complementarity determining region CDR1 is shown in SEQ ID NO. 7; the amino acid sequence of the amino acid sequence complementarity determining region CDR2 is shown in SEQ ID NO. 8; the amino acid sequence of the amino acid sequence complementarity determining region CDR3 is shown in SEQ ID NO 9.

2. The nanobody of claim 1, which has the amino acid sequence shown in SEQ ID NO. 1.

3. The nanobody for specifically recognizing Vibrio parahaemolyticus according to claim 2, wherein the nucleotide sequence encoding the nanobody is represented by SEQ ID NO 2.

4. A detection kit for specifically recognizing Vibrio parahaemolyticus, comprising the nanobody of any one of claims 1 to 3 for specifically recognizing Vibrio parahaemolyticus.

5. A recombinant vector comprising the nucleotide sequence of claim 3 encoding said nanobody.

6. A host cell comprising the recombinant vector of claim 5.