CN108864281B - A nanobody against Salmonella enteritidis and its application - Google Patents

Abstract

The present invention relates to an anti-Salmonella enteritidis nanobody and its application. The antibody has the function of binding to Salmonella Enteritidis. The present invention discloses the nanobody, the gene sequence encoding the nanobody, the expression vector and host cell of the nanobody, and the method for producing the nanobody. The anti-Salmonella Enteritidis nanobody of the present invention has the advantages of small size, high expression efficiency, good solubility, strong stability and the like. The present invention also includes the use of nanobodies against Salmonella Enteritidis.

Description

A nanobody against Salmonella enteritidis and its application

technical field

The invention relates to the fields of molecular biology, phage display technology and proteomics, in particular to an anti-Salmonella Enteritidis nanobody and its application.

Background technique

Salmonella is a gram-negative short bacillus, and there are more than 1,000 kinds of bacteria. It can cause animal diseases such as livestock, mice and poultry. Once it contaminates human food, it will cause human food poisoning. Salmonella mainly includes A. paratyphi, B. paratyphoid, B. typhimurium, C. paratyphi, Bacillus cholerae, Bacillus typhi and Enteritidis. Among them, Salmonella Enteritidis is highly invasive, mainly polluting meat, eggs, poultry and aquatic products, and can cause human enteritis and food poisoning. According to statistics, in developed countries such as Japan and the United States, nearly 80% of food poisoning incidents are caused by Salmonella Enteritidis, which has caused serious impact on human health and social economy.

The traditional detection method of Salmonella Enteritidis is culture method. After enrichment of samples, isolation and culture are carried out, and biochemical tests and serological identification of suspicious colonies are carried out. The culture method is currently the gold standard for detection of Salmonella in countries around the world. However, this method has the disadvantage of long detection time, and it often takes 4 to 7 days to obtain the detection results, which cannot meet the requirements of rapid screening. Therefore, the establishment of a rapid and accurate detection method for Salmonella Enteritidis is of great importance for ensuring food safety and consumers. health is important.

Immunoassay is an analysis method based on antigen-antibody reaction, which has the advantages of high detection sensitivity, simple operation and low cost. The core of immunoassay methods is antibodies. Currently, there are monoclonal and polyclonal antibodies against Salmonella Enteritidis on the market. However, polyclonal antibodies have the disadvantage of poor uniformity. The production process of monoclonal antibodies is complicated, the cycle is long, and the cost is high. There is an urgent need for an antibody with good specificity, low production cost and stable performance. A class of antibodies lacking light chains naturally exists in camelid animals, which are called heavy chain antibodies. Single-domain heavy chain antibodies can be obtained by cloning and expressing heavy chain antibodies. Because of their small size (only about 17KD), they are also called nanobodies. . Nanobody is a kind of genetically engineered antibody, which has the advantages of low production cost, high expression efficiency, easy genetic modification, good stability, etc., and has very good application prospects. However, no nanobody against Salmonella Enteritidis has yet been reported. Therefore, there is an urgent need to develop effective anti-Salmonella Enteritidis nanobodies.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an effective anti-Salmonella enteritidis nanobody and its application.

An anti-Salmonella Enteritidis nanobody, the amino acid sequence framework region of the nanobody includes FR1, FR2 and FR3, and the complementary variable region includes CDR1, CDR2 and CDR3;

The FR1 is selected from the following sequence: the sequence shown in SEQ ID NO: 3; the sequence with more than 90% homology with SEQ ID NO: 3; the FR2 is selected from the following sequence: SEQ ID NO: 3 The sequence shown in ID NO: 4; the sequence with more than 90% homology with SEQ ID NO: 4; the FR3 is selected from the following one sequence: the sequence shown in SEQ ID NO: 5; with SEQ ID NO: 5 sequences with more than 90% homology;

The CDR1 is selected from the sequence of the following one: the sequence shown in SEQ ID NO: 6; the sequence with more than 90% homology with SEQ ID NO: 6; the CDR2 is selected from the sequence of the following one: SEQ ID NO: 6 The sequence shown in ID NO: 7; the sequence with more than 90% homology with SEQ ID NO: 7; the CDR3 is selected from the sequence of one of the following: the sequence shown in SEQ ID NO: 8; and the sequence shown in SEQ ID NO: 8 Sequences with more than 90% homology.

Optionally, the amino acid sequence of the Nanobody is selected from one of the following sequences: the sequence shown in SEQ ID NO: 1; the sequence having more than 90% homology with SEQ ID NO: 1.

Optionally, the DNA sequence of the Nanobody is selected from one of the following sequences: the sequence shown in SEQ ID NO:2; the sequence with more than 90% homology with SEQ ID NO:2.

The application of the anti-Salmonella Enteritidis nanobody of the present invention for detecting Salmonella Enteritidis in food.

Optionally, the food includes drinking water, milk, beverages and fruit juice.

A Salmonella Enteritidis detection kit, which contains the anti-Salmonella Enteritidis nanobody of the present invention.

A recombinant carrier, which carries the anti-Salmonella Enteritidis nanobody of the present invention.

A host cell containing the anti-Salmonella Enteritidis nanobody of the present invention.

A method for producing an anti-Salmonella enteritidis nanobody, comprising culturing the host cell, obtaining a culture solution containing the anti-Salmonella enteritidis nanobody, and separating and purifying the anti-Salmonella enteritidis nanobody from the culture solution .

A polynucleotide encoding the amino acid sequence of the anti-Salmonella nanobody.

The beneficial effects of the present invention are:

(1) The anti-Salmonella Enteritidis nanobody provided by the present invention has a unique variable region sequence, so that the antibody has specific recognition and binding ability to Salmonella Enteritidis.

(2) The anti-Salmonella Enteritidis nanobody provided by the present invention has the advantages of easy expression and high expression efficiency.

(3) The anti-Salmonella Enteritidis nanobody provided by the present invention has the advantages of high affinity and strong specificity.

(4) The anti-Salmonella Enteritidis nanobody provided by the present invention has the advantage of strong stability.

(5) Based on the anti-Salmonella Enteritidis nanobody provided by the present invention, a rapid detection method for Salmonella Enteritidis in food can be established.

Description of drawings

Fig. 1 is a first round PCR amplification VHH gene electrophoresis identification diagram;

Fig. 2 is a second round PCR amplification VHH gene electrophoresis identification diagram;

Figure 3 is a comparison diagram of the phage-displayed Nanobody NB13;

Figure 4 is an SDS-PAGE electrophoresis image of the anti-Salmonella Enteritidis nanobody NB13;

Figure 5 shows the specificity of the anti-Salmonella Enteritidis Nanobody NB13;

Figure 6 shows the thermal stability of anti-Salmonella Enteritidis Nanobody NB13;

Figure 7 is a standard inhibition curve of Salmonella Enteritidis in Nanobody-based foods.

Detailed ways

Glossary:

Nanobodies: variable regions of camelid heavy chain antibodies;

Amino acid sequence framework region FR1, the first constant region sequence of the Nanobody;

The amino acid sequence framework region FR2, the second constant region sequence of the Nanobody;

Amino acid sequence framework region FR3, the third constant region sequence of the Nanobody;

Amino acid sequence complementarity determining region CDR1, the first variable region sequence of the Nanobody;

Amino acid sequence complementarity determining region CDR2, the second variable region sequence of the Nanobody;

Amino acid sequence complementarity determining region CDR3, the third variable region sequence of the Nanobody;

The anti-Salmonella Enteritidis nanobody of the present invention is used for the detection of Salmonella Enteritidis in foods, such as drinking water, milk, beverages, fruit juices and other liquid foods.

1. Construction of anti-Salmonella Enteritidis phage-displayed nanobody library

1.1 Immunization of camels: Select a camel that has not been immunized with any antigen, emulsify the inactivated Salmonella Enteritidis and Freund's complete adjuvant in a ratio of 1:1, and inject 10 ⁶ cfu/mL subcutaneously at multiple points. The camels were immunized by means of immunization, boosting immunization once every 2 weeks, and the subsequent immunization was replaced by incomplete Freund's adjuvant and inactivated Salmonella Enteritidis emulsified, a total of 5 times of immunization. One week after each immunization, blood was collected from camels to detect serum titers.

1.2 Extraction of total RNA from blood: after the fifth immunization, peripheral blood of camel was taken, and lymphocytes were separated from the blood according to routine procedures in the art to extract total RNA.

1.3 Reverse transcription to obtain cDNA:

The obtained total RNA was used as a template, and oligo(dT) ₁₅ was used as a primer for reverse transcription to synthesize the first strand of cDNA to obtain a cDNA library.

1.4 Amplification of Nanobody (VHH) Gene Fragment:

The first round of PCR amplification was performed with the synthesized cDNA as template and CALL001 and CALL002 as primers. The reaction system is as follows:

Vortex to mix, and after a brief centrifugation, the PCR amplification reaction is performed. The PCR conditions are:

(1)94℃2min;

(2)94℃30s;

(3)55℃30s;

(4)68℃1min;

(2)～(4) 30 cycles of amplification;

(5) 5min at 68°C.

In above-mentioned scheme, described PCR amplification VHH its forward primer CALL001 is:

5’-GTCCTGGCTGCTCTTCTACAAGG-3’

The reverse primer CALL002 is:

CALL002:5'-GGTACGTGCTGTTGAACTGTTCC-3'

After the PCR products were separated by 1% agarose gel electrophoresis, the DNA fragments of 700bp in size were purified and recovered by the kit, which was the first round of PCR amplification of the VHH gene. The specific electrophoresis identification diagram is shown in Figure 1, and M in the figure represents DL 2000 marker, 1 represents the first round of PCR amplification of VHH gene products.

The second round of PCR amplification was carried out using the first round of PCR amplification of VHH gene products as templates and CAM-FOR and CAM-BACK as primers.

The reaction system is as follows:

Vortex to mix, and after a brief centrifugation, the PCR amplification reaction is performed. The PCR conditions are:

(1)94℃2min;

(2)94℃30s;

(3)55℃30s;

(4)68℃1min;

(2)～(4) 20 cycles of amplification;

(5) 5min at 68°C.

In above-mentioned scheme, described PCR amplification VHH its forward primer CAM-FOR is:

CAM-FOR: 5'-GGCCCAGGCGGCCGAGTCTGGRGGAGG-3'

The reverse primer CAM-BACK is:

CAM-BACK: 5’-GGCCGGCCTGGCCGGAGACGGTGACCAGGGT-3’

After the PCR product was separated by 1% agarose gel electrophoresis, a DNA fragment of 400bp in size was purified and recovered by a kit, which is the VHH fragment. The specific electrophoresis diagram is shown in Figure 2. In Figure 2, M represents the DL 2000 marker, and 1 represents the second Round PCR amplification of the VHH gene product.

1.4 Construction of the vector

Digestion of pComb3xss:

The reaction solution was prepared according to the following system:

After the digestion product was separated by 1% agarose gel electrophoresis, a 3400bp vector fragment was purified and recovered with a kit.

Ligation of VHH gene with double-digested pComb3xss vector

In-Fusion connection is made according to the following system:

React overnight at 16°C for 16 hours, recover with agarose gel DNA purification kit, and store at -20°C until use. 1.5 Electrotransformation of ligation products

Add 3 μL of the above ligation product to 50 μL of E.coli ER2738 electrotransformation competent cells, after mixing, add it to a pre-cooled 0.1 cm electroporation cup (Bio-RAD), and then place it in a Bio-rad electroporation instrument. Electrotransformation was carried out on the electrotransformation condition: 1.8kV, 200Ω, 25μF. Immediately after the electrotransformation, 1mL of preheated SOC liquid medium was added to the electroconversion cup, and then transferred to a clean sterilized 15mL shaker tube after pipetting. As described above, ten times of electroporation were performed, and the bacterial solution after ten times of electroporation was mixed, and slowly shaken at 37°C for 1 h.

1.6 Construction of anti-Salmonella Enteritidis phage-displayed nanobody library

Transfer all the recovered bacterial liquid into 200 mL of SB medium, shake at 250 rpm at 37 °C until the OD ₆₀₀ value is 0.5, add 1 mL of 1 × 10 ¹² pfu helper phage M13KO7, stand at 37 °C for 1 h, and continue to shake. Shake for 2 h, add kanamycin to a final concentration of 70 μg/mL, and shake overnight. The next day, the overnight bacteria were centrifuged at 10000 rpm at 4°C for 15 min, the supernatant was transferred to a sterile centrifuge bottle, 1/4 volume of 5x PEG/NaCl was added, and after standing on ice for 2 h, centrifugation at 12000 rpm at 4°C for 20 min, The amplified anti-Salmonella Enteritidis phage-displayed nanobody library was obtained by dissolving the pellet with 10 mL of sterile resuspension solution (PBS buffer containing 1× protease inhibitors, 0.02% NaN ₃ and 0.5% BSA).

2. Panning and identification of anti-Salmonella Enteritidis Nanobodies

2.1 Panning of anti-Salmonella Enteritidis Nanobodies:

The inactivated Salmonella Enteritidis was coated on the ELISA plate, and after blocking with 3% nonfat milk powder, 100 μL of the phage-displayed nanobody library was added to each well, and it was left standing at 37°C for 2 h, the supernatant was discarded, and the plate was washed with 0.05% PBST solution. Six times, 100 μL of glycine solution (pH 2.2) was added to elute the adsorbed phage-displayed Nanobodies. After amplifying the eluted phage, a second round of panning was performed, and the panning process was the same as that of the first round of panning. Four rounds of panning were thus performed, and after each panning, the titers of eluted and added phage-displayed Nanobodies were determined.

2.2 Identification of Anti-Salmonella Enteritidis Nanobodies:

The panned phage-displayed Nanobodies were identified by ELISA. The specific operation is as follows: coat the ELISA plate with a certain concentration of inactivated Salmonella Enteritidis, block it with 3% nonfat dry milk, add 100 μL of phage supernatant, let stand at 37°C for 1 h, discard the supernatant, and use 0.05% PBST The solution was washed 6 times, 100 μL of enzyme-labeled anti-M13 secondary antibody was added, and incubated at 37°C for 1 h. After washing the plate, add TMB substrate for color development, incubate for 15 min, add stop solution, and read the OD value of each well with a microplate reader.

By direct ELISA, a phage-displayed nanobody that can bind to Salmonella Enteritidis was selected. As shown in Figure 3, the phage-displayed nanobody NB13 that can bind strongly to Salmonella Enteritidis is a positive phage, and it will be sent to a sequencing company for gene sequencing Sequencing. Use Bioedit software to analyze the sequencing results, log on the IMGT website (www.http://www.imgt.org/), analyze the antibody gene sequence, and determine the framework region and complementarity determining region of the antibody sequence.

The amino acid sequence of the amino acid sequence framework region FR1 of the anti-Salmonella Enteritidis Nanobody NB13 is shown in SEQ ID NO: 3;

The amino acid sequence of the amino acid sequence framework region FR2 of the anti-Salmonella Enteritidis Nanobody NB13 is shown in SEQ ID NO: 4;

The amino acid sequence of the amino acid sequence framework region FR3 of the anti-Salmonella Enteritidis Nanobody NB13 is shown in SEQ ID NO: 5;

The amino acid sequence of the amino acid sequence complementarity determining region CDR1 of the anti-Salmonella Enteritidis Nanobody NB13 is shown in SEQ ID NO: 6;

The amino acid sequence of the amino acid sequence complementarity determining region CDR2 of the anti-Salmonella Enteritidis Nanobody NB13 is shown in SEQ ID NO: 7;

The amino acid sequence of the amino acid sequence complementarity determining region CDR3 of the anti-Salmonella Enteritidis Nanobody NB13 is shown in SEQ ID NO: 8;

The amino acid sequence of the anti-Salmonella Enteritidis Nanobody NB13 is shown in SEQ ID NO: 1;

The DNA sequence of the anti-Salmonella Enteritidis Nanobody NB13 is shown in SEQ ID NO:2.

3. Expression of Anti-Salmonella Enteritidis Nanobodies

The phage-displayed nanobody NB13 plasmid was extracted and transformed into host cells TOP10F' by heat shock. The TOP10F' cells containing the nucleotide sequence of the nanobody NB13 were cultured overnight, and the next day, the cells were precipitated, the cell wall was disrupted by ultrasonication, and the nanobody was purified through a nickel column. The purification effect was identified by SDS-PAGE, and the results are shown in Figure 4. In Figure 4, M represents the takarapremix protein marker; in Figure 4, 1 represents the nanoanti-Salmonella Enteritidis nanobody NB13. The nanobody concentration was measured by the Bradford method, and the expression efficiency of the nanobody was calculated to be 5 mg/L medium.

4. Specificity Analysis of Anti-Salmonella Enteritidis Nanobodies

The interaction of anti-Salmonella Enteritidis nanobody NB13 with 5 different food-borne pathogens was determined by ELISA. Five pathogenic bacteria, Salmonella enteritidis, Salmonella typhimurium, Enterobacter sakazakii, Staphylococcus aureus, and Escherichia coli were coated on the ELISA plate. After blocking, 100 μL of nanobody NB13 solution was added, incubated at 37°C for 1 h, and washed with PBST. After plate three times, add anti-HA-HRP secondary antibody, let stand at 37°C for 1h, wash the plate six times with PBST, add TMB solution for color development for 15min, add sulfuric acid solution to stop the reaction, and judge the nanometer by measuring the absorbance value of each well at 450nm. specificity of the antibody. The results are shown in Figure 5, only the OD value of the well coated Salmonella Enteritidis is higher, and the OD values of other wells are comparable to the blank, indicating that the nanobody NB13 of the present invention has strong specificity to Salmonella Enteritidis.

V. Thermal stability analysis of anti-Salmonella Enteritidis Nanobodies

After the Salmonella Enteritidis nanobodies were incubated at 70°C for 0.5h, 1h, 1.5h and 2h, the activity changes of the nanobodies were detected by ELISA and compared with those of the non-heat-treated nanobodies. The results are shown in Figure 6, the nanobody can still maintain 60% activity after being treated at 70°C for 1 h, indicating that the nanobody has high thermal stability.

6. Establishment of a detection method for Salmonella Enteritidis in food

According to the screening pairing, the anti-Salmonella Enteritidis monoclonal antibody 2B4 (published by CN107688094A) was selected as the capture antibody, and NB13-HRP was used as the detection antibody to detect Salmonella Enteritidis by double-antibody sandwich immunoassay. The capture antibody 2B4 was coated on a 96-well microtiter plate, the coating concentration of each well was 10 μg/mL, overnight at 4 °C; the next day, the supernatant was discarded, and the plate was washed three times with 0.05% PBST, and then washed with 3 % skimmed milk powder was used to block each well, and 20-10 ⁸ CFU/mL Salmonella Enteritidis solution was prepared respectively. 50 μL of standard bacterial solution and 50 μL of detection antibody (ie, nanobody NB13) were added to each well, and incubated at 37° C. for 1 h. Wash the plate six times with 0.05% PBST, add TMB substrate color developing solution, develop color at room temperature for 15 min, add 2M sulfuric acid solution to stop the reaction, detect the OD value of each well at 450 nm, and draw a standard curve, the standard curve is shown in Figure 7 . The detection limit of this method was 10 ⁴ CFU/mL.

Embodiment 1. Detection of Salmonella Enteritidis in milk

The milk was purchased from the market, and after identifying the absence of Salmonella Enteritidis by the plate counting method, 1 CFU/mL of Salmonella was added, and after 24 hours of enrichment, the nanobody NB13 of the present invention was used for detection by the established double-antibody sandwich enzyme-linked immunosorbent assay method. . It can be seen from Table 1 that the established ELISA method can detect Salmonella Enteritidis in milk samples after 24h enrichment.

Table 1 Detection of Salmonella Enteritidis in enriched milk by enzyme-linked immunosorbent assay

Nucleotide sequence listing electronic file

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<120> An anti-Salmonella Enteritidis nanobody and its application

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<212> PRT

<213> Bactrian camel

<220> Amino acid sequence of anti-Salmonella Enteritidis Nanobody

<400>1

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

Gly Phe Pro Ser Ser Asp Ile Cys Met Gly

Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg

Glu Arg Val Ala Ala Ile Thr Ser Glu Gly

Tyr Thr Ser Ile Ala Asp Ser Val Lys Gly

Arg Phe Thr Ile Ser Gln Asp Lys Ala Lys

Asn Thr Leu Asp Leu Leu Met Asn Asn Leu

Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys

Ala Ala His Arg Gly Ala Trp Cys Tyr His

Ala Pro Arg Leu Phe Asn Phe Trp Gly Gln

Gly Thr Gln Val Thr Val Ser

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<213> Bactrian camel

<220> Nucleotide sequence of anti-Salmonella Enteritidis Nanobody

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gagtctggaggaggctcggtgcaggctggagggtctctgag

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gcatgggctggttccgccaggctccagggaaggagcgcgag

agagtcgcggctattactagtgaaggttacacaagcatcgc

agactccgtgaagggccgattcaccatctcccaagacaagg

ccaagaacactcttgatctactaatgaacaatttgaaacct

gaggacactgccatgtactactgtgcggcccatcgaggagc

ttggtgttatcacgcaccgcggctgtttaatttctggggcc

aggggacccaggtcaccgtctcc

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<211>20

<212> PRT

<213> Bactrian camel

<220> Amino acid sequence framework region FR1 of anti-Salmonella Enteritidis Nanobody

<400>3

Glu Ser Gly Gly Gly Ser Val Gln Ala Gly

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser

<210>4

<211>16

<212> PRT

<213> Bactrian camel

<220> Amino acid sequence framework region FR2 of anti-Salmonella Enteritidis Nanobody

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Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg

Glu Arg Val Ala Ala Ile

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<211>37

<212> PRT

<213> Bactrian camel

<220> Amino acid sequence framework region FR3 of anti-Salmonella Enteritidis Nanobody

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Ala Asp Ser Val Lys Gly Arg Phe Thr Ile

Ser Gln Asp Lys Ala Lys Asn Thr Leu Asp

Leu Leu Met Asn Asn Leu Lys Pro Glu Asp

Thr Ala Met Tyr Tyr Cys Ala

<210>6

<211>10

<212> PRT

<213> Bactrian camel

<220> Amino acid sequence complementarity determining region CDR1 of anti-Salmonella Enteritidis Nanobody

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Gly Phe Pro Ser Ser Asp Ile Cys Met Gly

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<212> PRT

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Thr Ser Glu Gly Tyr Thr Ser Ile

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<212> PRT

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<220> Amino acid sequence complementarity determining region CDR3 of anti-Salmonella Enteritidis Nanobody

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Pro Arg Leu Phe Asn Phe Trp Gly Gln Gly

Thr Gln Val Thr Val Ser

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<220> PCR amplification of VHH forward primer CALL001

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5’-GTCCTGGCTGCTCTTCTACAAGG-3’

<210>10

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<213> Synthetic

<220> PCR amplification of VHH reverse primer CALL002

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5’-GGTACGTGCTGTTGAACTGTTCC-3’

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<212> DNA

<213> Synthetic

<220> PCR amplification of VHH forward primer CAM-FOR

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5’-GGCCCAGGCGGCCGAGTCTGGRGGAGG-3’

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5'-GGCCGGCCTGGCCGGAGACGGTGACCAGGGT-3'.

Claims (5)

1. An anti-Salmonella Enteritidis Nanobody, wherein the amino acid sequence of the Nanobody is: the sequence shown in SEQ ID NO:1. 2. Application of the anti-Salmonella Enteritidis Nanobody of claim 1 for detection of Salmonella Enteritidis in food. 3. The application according to claim 2, wherein the food comprises drinking water, milk and fruit juice. 4. A Salmonella Enteritidis detection kit, characterized in that, the kit contains the anti-Salmonella Enteritidis Nanobody of claim 1. 5 . A polynucleotide, characterized in that it encodes the amino acid sequence of the anti-Salmonella nanobody according to claim 1 . 6 .

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