CN112707963A - Nano antibody, recombinant vector, host cell for broad-spectrum recognition of salmonella and application thereof - Google Patents

Abstract

The invention discloses a broad-spectrum salmonella-resistant nano antibody, the amino acid sequence of which is shown as SEQ ID NO. 1, and also provides a vector and a host cell containing the nano antibody nucleotide sequence. The nano antibody can be well combined with salmonella enteritidis, salmonella typhimurium, salmonella hadamard, salmonella london and salmonella paratyphi B. At present, both the monoclonal antibody and the polyclonal antibody have poor stability and higher preparation cost, and can be easily combined with surface protein of staphylococcus aureus. The enzyme-linked immunoassay method of the double-nano antibody sandwich established by the invention can detect various salmonella, and the detection result is better. 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.

Description

Nano antibody, recombinant vector, host cell for broad-spectrum recognition of salmonella and application thereof

Technical Field

The invention belongs to the fields of molecular biology and immunoassay technology, and particularly relates to a nano antibody for broad-spectrum recognition of salmonella.

Background

Salmonella is a food-borne pathogen and is the leading cause of gastroenteritis, diarrhea and pain. Its infection can lead to high morbidity and mortality, especially in infants, the elderly or immunocompromised patients. Three million people die worldwide each year from salmonella infections, with salmonella serotype typhimurium and serotype enteritidis being the most prevalent. Given the low infectious dose of most food-borne pathogens, it is desirable to monitor the presence of food-borne pathogens at every step of food production, processing, distribution and storage. Therefore, the development of rapid and sensitive detection methods is crucial to ensure the safety of our food supply.

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. Furthermore, the bacterial population may be underestimated due to the presence of damaged or stressed cells, which may not be cultured on selective media. Immunological methods based on specific recognition between antibodies and antigens have been widely used for rapid detection of food-borne pathogenic bacteria, and antibodies, which are used as core elements in immunoassay methods, play a key role in specific recognition of antigens and sensitivity of detection methods. Conventional antibodies such as monoclonal antibodies or polyclonal antibodies have long preparation periods and high storage costs, and the titer of antibodies in different batches also varies, and moreover, the production of these antibodies does not meet the current trend of animal welfare, which has prompted 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.

In the current prior art, the applicant discloses a nano antibody against salmonella enteritidis and application thereof in patent CN108864281B filed on 7/13/2018 and finally granted, however, the nano antibody is mainly applied to salmonella enteritidis and has no identification capability for other types of salmonella.

The Chinese patent application CN109932505A provides a test strip for detecting a broad-spectrum salmonella in food, and discloses a monoclonal antibody SP1 and a polyclonal antibody DSP1 which can identify the broad-spectrum salmonella of 32 different salmonella serotypes, but does not disclose the amino acid or nucleotide sequence of the antibody.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a nano antibody which has good recognition and binding capacity on most salmonella such as salmonella enteritidis, salmonella typhimurium, salmonella hadamard, salmonella london, salmonella paratyphi B and the like, and the nano antibody is used as a capture antibody and a detection antibody and applied to the technologies such as a salmonella detection kit and the like, and the nano antibody is realized by the following technologies.

The amino acid sequence of the nano antibody for widely recognizing salmonella 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 (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 a broad-spectrum nano antibody, and has good binding capacity on salmonella enteritidis, salmonella typhimurium, salmonella hadamard, salmonella london and salmonella paratyphi B. The present monoclonal antibody and polyclonal antibody have poor stability and are easy to combine with surface protein of staphylococcus aureus, and the nano antibody provided by the invention has the advantages of good broad spectrum, strong stability, high expression yield and no cross reaction with staphylococcus aureus.

The enzyme-linked immunoassay method of the double-nano antibody sandwich established by the nano antibody can detect various salmonella, 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 the broad-spectrum identification of the salmonella is shown as SEQ ID NO. 1.

More preferably, the nucleotide sequence of the nano antibody is shown in 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 the broad-spectrum identification of salmonella, which contains the broad-spectrum identification salmonella nano antibody.

For exampleThe double-nano antibody sandwich enzyme-linked immunoassay detection kit is prepared by using the nano antibody as a detection reagent, and the salmonella is subjected to double-antibody sandwich enzyme-linked immunoassay broad-spectrum detection by using the anti-salmonella nano antibody as a capture antibody and the phage-displayed nano antibody as a detection antibody. The detection limit of the salmonella by adopting the kit is 5.08 multiplied by 10⁴CFU/mL。

The invention also provides a recombinant vector, wherein the recombinant vector contains the nucleotide sequence of the nano antibody; also provides a host cell, which contains the recombinant vector. The recombinant vector and the host cell can be used for preparing the nano antibody.

Compared with the prior art, the invention has the advantages that: the invention provides a broad-spectrum anti-salmonella nano antibody which has good binding capacity to salmonella enteritidis, salmonella typhimurium, salmonella hadamard, salmonella london and salmonella paratyphi B. The monoclonal antibody and the polyclonal antibody have poor stability and are easy to combine with surface protein of staphylococcus aureus at present, and the anti-salmonella nano antibody provided by the invention has the advantages of good broad spectrum, strong stability, high expression yield and no cross reaction with staphylococcus aureus. The enzyme-linked immunoassay method of the double-nano antibody sandwich established by the invention can detect various salmonella, and the detection result is better. 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.

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-Salmonella nanobody NB 422;

FIG. 5 is a thermal stability analysis of anti-Salmonella nanobody NB 422;

FIG. 6 is a specific analysis of anti-Salmonella nanobody NB 422;

FIG. 7 is a standard curve of the anti-Salmonella nano-antibody NB422 for the detection of Salmonella enteritidis.

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 derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Examples

1. Construction of anti-salmonella phage display nano antibody library

1.1 camel immunization:

selecting a camel which is healthy and is not immunized with any antigen, priming the bactrian camel with an emulsified mixture of inactivated salmonella enteritidis and Freund's complete adjuvant in the same volume at the concentration of 106cfu/mL, emulsifying the antigen with Freund's incomplete adjuvant in the subsequent immunization, and boosting the immunization for 1 time every 2 weeks for 5 times in total. 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, after short-time centrifugation, carrying out 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 (shown in SEQ ID NO:11) is:

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 DL 2000marker, 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’-GGCCCAGGCGGCCGAGTCTGGRGGAGG-3’

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

CAM-BACK:5’-GGCCGGCCTGGCCGGAGACGGTGACCAGGGT-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 DL 2000marker, 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

ddH2O to the Total 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

ddH2O to the Total 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

Thawing 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 anti-Salmonella phage display Nanobody library

Transferring the recovered bacteria liquid into 200mL SB culture medium, and shaking at 37 deg.C and 250rpm to OD₆₀₀When the value is 0.5, 1mL of 1X 10 solution is added¹¹pfu 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, the mixture was allowed to stand on ice for 2h, 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-Salmonella phage display Nanobody library.

2. Elutriation and identification of anti-salmonella nano antibody

2.1 panning of anti-salmonella nanobodies:

inactivated Salmonella enteritidis is used as a coating antigen, each well is 100 mu L, and the concentration is 10⁸CFU/ml (round-by-round decrease in the concentration of the coated bacteria), adding 300. mu.L of 3% skimmed milk powder to block for 1h, washing the plate 3 times with PBST solution containing 0.05%, Tween-20, adding 100. mu.L of phage display nanobody library to each well, incubating at 37 ℃ for 1h, washing the plate 6 times, adding 100. mu.L of glycine solution (pH 2.2) to elute, immediately adding Tris-HCL neutralizes 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 round, the third round and the fourth round of elutriation processes, the concentration of the coating antigen is 5 multiplied by 10 in sequence⁷CFU/ml、1×10⁷CFU/ml and 5X 10⁶CFU/ml, the remaining panning process was the same as the first panning, for a total of 4 panning rounds. After the fourth panning, 10 μ L phage was used 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 by indirect ELISA for positive clones.

2.2 identification of anti-salmonella nanobody:

and carrying out positive clone identification on the panned phage display nano antibody by indirect ELISA. The specific operation is as follows: coating with inactivated Salmonella enteritidis with a certain concentration overnight at 4 ℃, sealing with 3% skimmed milk powder, adding 100 μ L phage-displayed nano antibody, standing at 37 ℃ for 1h, discarding the supernatant, washing the plate with 0.05% PBST solution for 6 times, adding 100 μ L enzyme-labeled anti-M13 secondary antibody, and incubating at 37 ℃ for 1 h. Washing the plate for 6 times, adding TMB substrate for color development, incubating for 15min, adding 50 μ L H₂SO₄The 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, the nano antibody with higher affinity can be panned, positive clone which can be combined with salmonella enteritidis is obtained through indirect ELISA screening, sequencing results are analyzed by Bioedit software, IMGT website (www.http:// www.imgt.org /) is logged, the gene sequence of the antibody is analyzed, and the framework region and the complementary determining region of the antibody sequence are determined.

3. Preparation of anti-salmonella nano antibody

3.1 preparation of phage-displayed Nanobody phage422 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 phage422, 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 NB422 by protein expression

The plasmid of the phage display nano antibody phage422 is extracted and transformed into expression strain TOP 10F' by heat shock. 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-salmonella nanobody NB 422. The concentration of the nano antibody is measured by using the Nanodrop, and the yield of the nano antibody is 6.5mg/L of culture medium through calculation.

The amino acid sequence of the amino acid sequence framework region FR1 of the anti-salmonella nano antibody NB422 is shown as SEQ ID NO. 3;

the amino acid sequence of the amino acid sequence framework region FR2 of the anti-salmonella nano antibody NB422 is shown as SEQ ID NO. 4;

the amino acid sequence of the amino acid sequence framework region FR3 of the anti-salmonella nano antibody NB422 is shown as SEQ ID NO. 5;

the amino acid sequence of the amino acid sequence framework region FR4 of the anti-salmonella nano antibody NB422 is shown as SEQ ID NO. 6;

the amino acid sequence of the complementary determining region CDR1 of the amino acid sequence of the anti-salmonella nano antibody NB422 is shown as SEQ ID NO. 7;

the amino acid sequence of the complementary determining region CDR2 of the amino acid sequence of the anti-salmonella nano antibody NB422 is shown as SEQ ID NO. 8;

the amino acid sequence of the complementary determining region CDR3 of the amino acid sequence of the anti-salmonella nano antibody NB422 is shown as SEQ ID NO. 9;

the amino acid sequence of the anti-salmonella nano antibody NB422 is shown in SEQ ID NO. 1;

the nucleotide sequence of the anti-salmonella nano antibody NB422 is shown in SEQ ID NO. 2.

4. Anti-salmonella nano-antibody thermal stability analysis

Coating inactivated salmonella enteritidis with certain concentration on an enzyme label plate, coating overnight at 4 ℃, washing the plate for 3 times in 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 2M H for developing₂SO₄The reaction was stopped and the absorbance was measured at 450 nm. Comparing the influence of different temperature treatments on the activity of the nano antibody, the results are shown in fig. 5, the nano antibody can keep better antigen binding ability at 37-70 ℃, and can still keep 50% of activity after being treated at 80 ℃ for 15min, which indicates that the nano antibody has certain thermal stability.

5. Specificity analysis of anti-salmonella nano antibody

The binding capacity of the nano antibody NB422 and other food-borne pathogenic bacteria is determined by using a double nano antibody sandwich method and 5 kinds of salmonella and 5 kinds of non-salmonella as analytes. Coating 20 mu g/mL of anti-salmonella nano antibody on an enzyme label plate at 4 ℃, adding 300 mu L of skimmed milk powder into each hole for sealing for 1h the next day, washing the plate for 3 times, adding 5 kinds of salmonella of enteritis salmonella, salmonella typhimurium, salmonella hadamari, salmonella london, salmonella paratyphi B and staphylococcus aureus, candida albicans, campylobacter jejuni and escherichia coli O157 with certain concentrations: h7, 5 non-salmonella of Enterobacter sakazakii, incubating for 1H, washing the plate for 3 times, adding 100 mu L of phage display nano antibody, incubating for 1h at 37 ℃, after washing the plate for three times by PBST, adding anti-M13-HRP secondary antibody, standing for 1h at 37 ℃, washing the plate for six times, adding TMB solution for developing for 15min, adding sulfuric acid solution to stop the reaction, and determining the specificity of the nano antibody by measuring the absorbance value of each hole under 450 nm. As a result, as shown in FIG. 6, the Nanobody NB422 cross-reacted with 5 kinds of Salmonella and the OD values were comparable. The nanobody NB422 did not bind to other 5 non-salmonella bacteria. The experimental result shows that the nano antibody NB422 can be used for identifying 5 kinds of salmonella in a broad spectrum manner, and is strong in binding capacity and good in broad spectrum.

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

According to screening pairing, an anti-salmonella nano antibody NB422 is selected as a capture antibody, and a nano antibody phase 422 displayed by a phage is selected as a detection antibody to carry out double-antibody sandwich enzyme-linked immunoassay to detect salmonella. The method comprises the following specific steps: coating the capture antibody NB422 on a 96-well enzyme label plate, wherein the coating concentration of each well is 20 mug/mL, and the temperature is 4 ℃ overnight; the next day, the supernatant was discarded, the plates were washed three times with 0.05% PBST, then blocked with 3% skimmed milk for 1h, and Salmonella enteritidis was diluted in a gradient at a concentration of 1.69X 10⁴～3.33×10⁸CFU/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 μ L phage display nano antibody phage422, incubating for 1h at 37 ℃, washing the plate three times, adding anti-M13 enzyme-labeled secondary antibody, 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 5.08 multiplied by 10⁴CFU/mL。

Sequence listing

<110> northwest agriculture and forestry science and technology university

<120> nano antibody for identifying salmonella in broad spectrum, recombinant vector, host cell and application thereof

<160> 13

<170> SIPOSequenceListing 1.0

<210> 2

<211> 127

<212> PRT

<213> camel (Bactrian camel)

<400> 2

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

1 5 10 15

Cys Ala Val Ser Arg Ala Thr Asp Thr Arg Tyr Cys Met Gly Trp Phe

20 25 30

Gln Pro Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Thr

35 40 45

Thr Lys Asn Ser Arg Gly Val Ser Thr Phe Tyr Gly Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Gln Asp Gly Ala Arg Asn Thr Met Ser Leu

65 70 75 80

Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Arg Val Pro Cys Gln Ser Met Thr Gly Lys Ala Thr Thr Leu Glu

100 105 110

Tyr Gly Tyr Asn Tyr Trp Gly Gln Gly Ile Gln Val Thr Val Ser

115 120 125

<210> 2

<211> 381

<212> DNA

<213> camel (Bactrian camel)

<400> 2

gagtctggag gagggtcggt gcaggctgga gggtctctga gactctcttg tgcagtttct 60

agagccacgg atactaggta ctgcatgggc tggttccgcc aggctccagg gaaggagcgc 120

gagggggtcg cagccattta tactactaag aatagccgag gtgttagcac attctatggc 180

gactccgtga agggccgatt caccatctcc caagacggcg ccaggaacac catgtctctg 240

caaatgaaca gcctgcaacc tgaagacact gccgtgtact actgcgcggc tcgagtgccc 300

tgtcagtcta tgacggggaa agcgaccaca ctggaatatg gatataacta ctggggacag 360

gggatccagg tcaccgtctc c 381

<210> 3

<211> 20

<212> PRT

<213> camel (Bactrian camel)

<400> 3

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

1 5 10 15

Cys Ala Val Ser

20

<210> 4

<211> 16

<212> PRT

<213> camel (Bactrian camel)

<400> 4

Trp Phe Gln Pro Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile

1 5 10 15

<210> 5

<211> 37

<212> PRT

<213> camel (Bactrian camel)

<400> 5

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

1 5 10 15

Thr Met Ser Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Val

20 25 30

Tyr Tyr Cys Ala Ala

35

<210> 6

<211> 10

<212> PRT

<213> camel (Bactrian camel)

<400> 6

Trp Gly Gln Gly Ile Gln Val Thr Val Ser

1 5 10

<210> 7

<211> 10

<212> PRT

<213> camel (Bactrian camel)

<400> 7

Arg Ala Thr Asp Thr Arg Tyr Cys Met Gly

1 5 10

<210> 8

<211> 14

<212> PRT

<213> camel (Bactrian camel)

<400> 8

Tyr Thr Thr Lys Asn Ser Arg Gly Val Ser Thr Phe Tyr Gly

1 5 10

<210> 9

<211> 20

<212> PRT

<213> camel (Bactrian camel)

<400> 9

Arg Val Pro Cys Gln Ser Met Thr Gly Lys Ala Thr Thr Leu Glu Tyr

1 5 10 15

Gly Tyr Asn Tyr

20

<210> 10

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gtcctggctg ctcttctaca agg 23

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ggtacgtgct gttgaactgt tcc 23

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggcccaggcg gccgagtctg grggagg 27

<210> 13

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggccggcctg gccggagacg gtgaccaggg t 31

Claims (6)

Translated fromChinese

1.广谱识别沙门氏菌的纳米抗体，其特征在于，所述纳米抗体的氨基酸序列包括氨基酸序列框架区FR1、FR2、FR3、FR4，以及氨基酸序列互补决定区CDR1、CDR2、CDR3；1. Nanobody for broad-spectrum recognition of Salmonella, characterized in that the amino acid sequence of the Nanobody comprises amino acid sequence framework regions FR1, FR2, FR3, FR4, and amino acid sequence complementarity determining regions CDR1, CDR2, CDR3;氨基酸序列框架区FR1的氨基酸序列如SEQ ID NO:3所示；氨基酸序列框架区FR2的氨基酸序列如SEQ ID NO:4所示；氨基酸序列框架区FR3的氨基酸序列如SEQ ID NO:5所示；氨基酸序列框架区FR4的氨基酸序列如SEQ ID NO:6所示；The amino acid sequence of the amino acid sequence framework region FR1 is shown in SEQ ID NO:3; the amino acid sequence of the amino acid sequence framework region FR2 is shown in SEQ ID NO:4; the amino acid sequence of the amino acid sequence framework region FR3 is shown in SEQ ID NO:5 ; The amino acid sequence of the amino acid sequence framework region FR4 is shown in SEQ ID NO: 6;氨基酸序列互补决定区CDR1的氨基酸序列如SEQ ID NO:7所示；氨基酸序列互补决定区CDR2的氨基酸序列如SEQ ID NO:8所示；氨基酸序列互补决定区CDR3的氨基酸序列如SEQID NO:9所示。The amino acid sequence of the amino acid sequence complementarity determining region CDR1 is as shown in SEQ ID NO:7; the amino acid sequence of the amino acid sequence complementarity determining region CDR2 is as shown in SEQ ID NO:8; the amino acid sequence of the amino acid sequence complementarity determining region CDR3 is as shown in SEQ ID NO:9 shown.2.根据权利要求1的广谱识别沙门氏菌的纳米抗体，其特征在于，所述纳米抗体的氨基酸序列如SEQ ID NO:1所示。2 . The nanobody for broad-spectrum recognition of Salmonella according to claim 1 , wherein the amino acid sequence of the nanobody is shown in SEQ ID NO: 1. 3 .3.根据权利要求2所述的广谱识别沙门氏菌的纳米抗体，其特征在于，所述纳米抗体的核苷酸序列如SEQ ID NO:2所示。3 . The nanobody for broad-spectrum recognition of Salmonella according to claim 2 , wherein the nucleotide sequence of the nanobody is shown in SEQ ID NO: 2. 4 .4.一种广谱识别沙门氏菌的检测试剂盒，其特征在于，含有权利要求1所述的广谱识别沙门氏菌纳米抗体。4. A detection kit for broad-spectrum identification of Salmonella, characterized in that it contains the broad-spectrum identification of Salmonella nanobody of claim 1.5.一种重组载体，其特征在于，所述重组载体上含有权利要求2所述的纳米抗体的核苷酸序列。5. A recombinant vector, characterized in that, the recombinant vector contains the nucleotide sequence of the Nanobody of claim 2.6.一种宿主细胞，其特征在于，所述宿主细胞内含有权利要求5所述的重组载体。6 . A host cell, characterized in that, the host cell contains the recombinant vector of claim 5 .

Images