Nano antibody for resisting salmonella enteritidis, kit and application of nano antibody

Abstract

The invention discloses a nano antibody for resisting salmonella enteritidis, a kit and application of the nano antibody, and belongs to the technical field of genetic engineering. The nano-antibody comprises at least one of the following: A1) a nano-antibody containing amino acid sequences of a complementary determining region CDR1 as shown in SEQ ID NO: 1, a complementary determining region CDR2 as shown in SEQ ID NO: 2 and a complementary determining region CDR3 as shown in SEQ ID NO: 3; a2) a nano-antibody containing a complementary determining region CDR1 shown as SEQ ID NO.6, a complementary determining region CDR2 shown as SEQ ID NO.7 and a complementary determining region CDR3 shown as SEQ ID NO.8. The nano-antibody for resisting salmonella enteritidis provided by the invention is strong in affinity and good in specificity, so that the nano-antibody has a relatively good application prospect in detection of salmonella enteritidis.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a nano antibody against enteritidis Salmonella, a kit and applications thereof. Background Art

[0002] Salmonella enteritidis is one of the main pathogens causing salmonellosis in piglets, especially in weaned piglets. The prevalence of salmonellosis in piglets is affected by many factors, including the age of the pig herd, hygiene conditions, feeding management, seasonal changes, and regional differences. Feed is one of the important transmission media of salmonellosis in piglets. The presence of Salmonella enteritidis in piglet feed is highly harmful and can cause paratyphoid fever in piglets, causing significant economic losses to the pig industry. In order to prevent the spread of Salmonella enteritidis in piglet feed, strict biosafety measures need to be taken, including quality control of feed raw materials, hygiene management during processing, optimization of storage conditions, and regular microbiological testing of feed. Conventional detection methods for Salmonella enteritidis include: pre-enrichment, selective enrichment, plate separation, biochemical tests, serological identification, polymerase chain reaction (PCR), real-time fluorescence quantitative PCR, etc. Although these analytical methods are highly accurate, the instruments they require are expensive, the sample separation, extraction, purification, derivatization and other pre-treatments are complex, the analysis speed is slow, the detection sensitivity is low, and it is difficult to achieve rapid on-site detection. With the rapid increase in the number of samples to be tested, especially those requiring rapid on-site detection, traditional analytical methods are difficult to adapt to the requirements. Therefore, it is urgent to develop an efficient and convenient method for detecting Salmonella Enteritidis.

[0003] Immunomagnetic bead analysis is a method for detecting microorganisms, proteins and other substances based on the specific reaction of antigens and antibodies. Compared with traditional instrument detection, it has the advantages of simple operation, high sensitivity, strong specificity and the ability to achieve rapid on-site detection. It can provide an efficient and convenient detection method for residual detection of Enteritidis Salmonella in feed. The selection of antibodies is the core of immunoassay methods. Compared with conventional antibodies, nanobodies (VHH) have the characteristics of small molecular weight, easy expression, and low production cost. Nanobodies also have the advantages of relatively simple preparation process and low antibody cross-reaction.

[0004] However, existing nanoantibodies used to detect Enteritidis Salmonella still have problems such as low sensitivity and poor specificity. Summary of the invention

[0005] The purpose of the present invention is to provide a nano antibody, a kit and an application thereof for resisting enteritidis Salmonella, so as to solve the problems of low sensitivity and poor specificity of the existing nano antibodies for detecting enteritidis Salmonella.

[0006] In a first aspect, the present invention provides an anti-Salmonella Enteritidis Nanobody, the Nanobody comprising at least one of the following: A1) a Nanobody comprising an amino acid sequence of a complementary determining region CDR1 as shown in SEQ ID NO: 1, a complementary determining region CDR2 as shown in SEQ ID NO: 2, and a complementary determining region CDR3 as shown in SEQ ID NO: 3; A2) a Nanobody comprising an amino acid sequence of a complementary determining region CDR1 as shown in SEQ ID NO: 6, a complementary determining region CDR2 as shown in SEQ ID NO: 7, and a complementary determining region CDR3 as shown in SEQ ID NO: 8.

[0007] The nanobodies (also called single-domain antibodies) provided by the present invention have only the variable region (VHH) of the heavy chain antibody, which is composed of the framework region FR1, the complementary determining region CDR1, the framework region FR2, the complementary determining region CDR2, the framework region FR3, the complementary determining region CDR3 and the framework region FR4 in sequence.

[0008] In the present invention, the term "nanobody" generally refers to an antibody consisting of only one H chain V region, which may also be referred to as a VHH antibody. The ability of a nanobody to bind to an antigen and its stability are substantially the same as those of a complete antibody. In this article, unless the context clearly indicates otherwise, when referring to the term "nanobody", it includes not only complete nanobodies, but also antigen-binding fragments of nanobodies. An antigen-binding fragment refers to a polypeptide comprising a fragment of a nanobody that retains the ability to specifically bind to the same antigen to which the nanobody binds, and / or competes with the nanobody for specific binding to the antigen, which is also referred to as an "antigen-binding portion".

[0009] In some embodiments, the amino acid sequence of a Nanobody is selected from any of the following: B1) having an amino acid sequence as shown in SEQ ID NO: 4 or SEQ ID NO: 9; B2) having one or more amino acid substitutions, deletions or additions compared to the amino acid sequence defined in B1) to obtain a functionally identical amino acid sequence; B3) an amino acid sequence that has more than 80% sequence identity and the same function as the amino acid sequence defined in B1) or B2); B4) an amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of B1).

[0010] In the present invention, the label includes but is not limited to: GST (glutathione sulfhydryl transferase) label protein, Trx (thioredoxin) label protein, nitrogen utilization substrate A (NusA) label protein, His label protein (His-tag), MBP (maltose binding protein) label protein, Flag label protein, SUMO label protein, HA (influenza hemagglutinin) label protein, Myc label protein, LacZ label protein, CBD (cellulose binding domain) label protein, bacteriophage T7 protein kinase (T7PK) label protein, GFP (green fluorescent protein), CFP9 (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag label protein. Those skilled in the art know how to select a suitable label protein according to the desired purpose. The use of the label does not change the function of the target protein (nanoantibody), and its purpose is to separate, purify, detect or trace, so the label protein suitable for this application is not limited to a specific type. The tag can be separated from the target protein (nanobody) by chemical cleavage methods or enzymatic methods known in the art (such as introducing a protease cleavage site and using TEV protease to cleave and remove the tag).

[0011] In a second aspect, the invention provides a nucleic acid molecule encoding any one of the Nanobodies described above.

[0012] The nucleic acid molecules provided by the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0013] In some embodiments, the nucleic acid molecule is selected from any of the following nucleic acid molecules: C1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 10; C2) a nucleic acid molecule that hybridizes under stringent conditions with the nucleic acid molecule defined in C1) and is any of the above-mentioned Nanobodies; C3) a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in C1) or C2) and encodes any of the above-mentioned Nanobodies.

[0014] As used herein, the term "hybridize under stringent conditions" means that two nucleic acid molecule fragments hybridize to each other under standard hybridization conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (1989) (Cold Spring Lane Laboratories, New York, USA) in the section "Expression of cloned genes in Escherichia coli". Such conditions are, for example, hybridization in 6.0×SSC at 45° C., followed by a washing step in 2×SSC at 50° C. In order to select stringency, the salt concentration in the washing step can be selected, for example, between 2.0×SSC at 50° C. for low stringency and 2.0×SSC at 50° C. for high stringency. In addition, the temperature in the washing step can be varied between room temperature of about 22° C. for low stringency and 65° C. for high stringency.

[0015] As used herein, the term "sequence identity" can be evaluated by the naked eye or by computer software (such as the software program described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology). When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. The identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. A "sequence identity" of a polynucleotide sequence or amino acid sequence to another sequence having a certain percentage (e.g., 90%, 95%, 98% or 99%) means that when the sequences are aligned, that percentage of bases or amino acids in the two sequences being compared are the same. In a third aspect, the present invention provides a recombinant vector comprising any one of the above-mentioned nucleic acid molecules.

[0016] The recombinant vector in the present invention includes a cloning vector and an expression vector. The cloning vector is used to replicate related sequences, and the expression vector is used to express related genes. For example, the expression vector in the present invention can be pET28a.

[0017] In a fourth aspect, the present invention provides a recombinant cell comprising any one of the above nucleic acid molecules or the above recombinant vectors.

[0018] In a fifth aspect, the present invention provides a method for preparing a nanobody against Enteritidis Salmonella, comprising the steps of: culturing the above-mentioned recombinant cells, inducing expression, and obtaining a culture; and isolating any one of the above-mentioned nanobodies from the culture.

[0019] In the present invention, there are no special requirements for the culture method and culture conditions, as long as the recombinant cells grow normally. And the methods for isolating the above-mentioned nanobodies from the culture are all conventional methods in the art.

[0020] In a sixth aspect, the present invention provides a kit for detecting Enteritidis Salmonella, comprising a capture antibody-magnetic bead complex and / or a detection antibody; wherein the capture antibody-magnetic bead complex is obtained by complexing the above-mentioned A1) nanoantibody with magnetic beads, the magnetic beads are ferroferric oxide particles containing primary amino groups on the surface, and the particle size of the ferroferric oxide particles is 180-220 nm; the detection antibody is a fusion protein obtained by fusing the above-mentioned A2) nanoantibody with alkaline phosphatase, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 11.

[0021] In some embodiments, the concentration of the capture antibody magnetic bead complex is 80-120 ng / mL, preferably 100 ng / mL; the concentration of the detection antibody is 0.1-0.5 μg / mL, preferably 0.3 μg / mL.

[0022] In some embodiments, the kit further comprises at least one of a Salmonella Enteritidis standard solution, a buffer solution, a washing solution, a color developing solution, and a reaction stopping solution.

[0023] In some preferred embodiments, the buffer is PBS buffer; the washing solution is PBST washing solution; the color developing solution is obtained by dissolving 15 mg of p-nitrophenyl phosphate (pNPP) in 15 mL of glycine buffer, and its pH value is 9.6; and the reaction termination solution is 3 M sodium hydroxide solution.

[0024] It is understandable that the kit also includes a box body and a detachable ELISA plate arranged in the box body.

[0025] In the seventh aspect, the present invention provides the use of any of the above-mentioned Nanobodies, any of the above-mentioned nucleic acid molecules, the above-mentioned recombinant vectors, the above-mentioned recombinant cells, the Nanobodies prepared by the above-mentioned preparation methods, and any of the above-mentioned kits in the detection of Enteritidis Salmonella and / or for the preparation of products for detecting Enteritidis Salmonella.

[0026] In the present invention, when the actual sample analysis and detection is performed, the capture antibody magnetic bead complex and the enteritis salmonella sample to be tested are added to each well of the sealed ELISA plate in sequence, and the sample is placed on a 96-well magnetic rack; the nano antibodies on the magnetic beads interact and bind with the enteritis salmonella to be tested, and then the detection antibody (fusion protein obtained by fusing the nano antibodies with alkaline phosphatase) is added to bind with the enteritis salmonella. Since the content of the magnetic beads and the detection antibody in each well is consistent, when the concentration of the enteritis salmonella to be tested is high, it is more bound to the coating antibody and the detection antibody, and finally the color developing solution and the reaction termination solution are added; the color developing reaction is deep, and the OD value detected by the ELISA instrument is high, indicating that the enteritis salmonella content in the sample is high; conversely, when the enteritis salmonella concentration to be tested is low, the measured OD value is low, indicating that the enteritis salmonella content in the sample is low. According to the standard curve drawn by detecting the enteritis salmonella standard solution with a known concentration, the concentration of the enteritis salmonella to be tested can be deduced.

[0027] The beneficial effects of the present invention are as follows: different from the prior art, the anti-Salmonella enteritidis nanoantibodies provided by the present invention have strong affinity and good specificity. When a kit containing the nanoantibodies is used to detect the content of Salmonella enteritidis in a sample, it has the advantages of good specificity and high sensitivity, and can detect samples quickly and in large quantities. Therefore, it has good application prospects in the detection of Salmonella enteritidis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is an SDS-PAGE image of the thiolated nanobody 1 prepared in Example 3 of the present invention, wherein lane M: marker, lane 1: thiolated nanobody 1; Figure 2 This is an SDS-PAGE image of the detection antibody (Nanobody 2 alkaline phosphatase fusion protein) prepared in Example 4 of the present invention, wherein lane M: marker, lane 1: Nanobody 2 alkaline phosphatase fusion protein; Figure 3 This is a standard curve diagram of Salmonella Enteritidis in Example 5 of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F. Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0031] Example 1 Construction of a Salmonella Enteritidis Phage Display Nanobody Library Salmonella Enteritidis (10 9 cfu / mL) was mixed with an equal volume of complete adjuvant (primary immunization) or Freund's incomplete adjuvant (secondary immunization and beyond), and after being fully emulsified, multiple injections were made into the muscles of the alpaca. After that, immunization was performed every 14 days, for a total of 6 immunizations. Seven days after the last immunization, peripheral blood lymphocytes of the alpaca were extracted, and total RNA was extracted. The VHH gene fragment was cloned by reverse transcription PCR and overlapping PCR, and restriction endonucleases were used to clone the VHH gene fragment. S I digested the pComb3X plasmid vector and scFv gene fragment, connected the VHH gene fragment to the phagemid pComb3x by T4 ligase, and efficiently electrotransformed it into Escherichia coli ER2738 to construct a phage nanobody library for Salmonella enteritidis. The primary library capacity was determined to be 10 8cfu / mL, and helper phage (infection multiplicity 20:1) M13KO7 was added for rescue to obtain a phage nanoantibody library (phage display VHH library) with a library capacity of 10 13 pfu / mL, and the diversity of the library was good.

[0032] In the reverse transcription PCR, the reverse transcription kit used was the M-MLV first-strand cDNA synthesis kit (purchased from OMEGA), and the reverse transcription system was shown in Table 1 below.

[0033] Table 1 Reverse transcription system

[0034] Reagents were prepared according to the reverse transcription system in Table 1, and reverse transcription was performed at 42°C for 30 min to obtain cDNA.

[0035] The cDNA obtained above was subjected to two rounds of overlapping PCR amplification, wherein the primer sequences (5′-3′) for PCR amplification are as follows: GSP-RT:CGCCATCAATRTACCAGTTGA(SEQ ID NO: 13); LP-leader:GTGGTCCTGGCTGCTCTW(SEQ ID NO: 14); F:CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC (SEQ ID NO: 15); R: CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG (SEQ ID NO: 16); Among them, R represents the base A / G, W represents the base A / T, and K represents the base G / T.

[0036] The reaction system of the first round of overlapping PCR is shown in Table 2 below.

[0037] Table 2 Reaction system

[0038] The reaction procedure for the first round of overlapping PCR is as follows: Pre-denaturation at 95°C for 3 min, 94°C for 30 s, 57°C for 30 s, 72°C for 45 s (25 cycles), extension at 72°C for 10 min, 4°C ∞.

[0039] The reaction system of the second round of overlapping PCR is shown in Table 3 below.

[0040] Table 3 Reaction system

[0041] The reaction procedure for the second round of overlapping PCR is as follows: Pre-denaturation at 95°C for 3 min, 94°C for 30 s, 57°C for 30 s, 72°C for 45 s (30 cycles), extension at 72°C for 10 min, 4°C ∞.

[0042] Example 2 Screening of Salmonella Enteritidis-specific Nanobodies The first well of a 96-well ELISA plate was coated with Salmonella Enteritidis at a concentration of 10 5 cfu / mL, 4°C overnight; the next day, pour out the coating solution, wash 3 times with PBST, add 150μL of 1% gelatin to the first two wells, add 150μL of 1% BSA solution to the last four wells, and incubate at room temperature for 1h; take 110μL of the phage display VHH library obtained in Example 1, add 110μL of 3% BSA solution, 25°C, 220rpm, shake on a shaker for 1h to allow the phage to fully bind to BSA, thereby removing non-specific phage antibodies; discard the liquid, wash three times with PBST, 1min each time, add 100μL of the premixed library to each of the first two wells, shake at room temperature for 2h, so that the phage can Fully bind to the coating source; discard the liquid, wash three times with PBST, 1 min each time; add 100µL of Gly-HCl buffer (0.2M, pH 2.2) and decolorize on a shaker for 10 min; then, add 20µL of Tris-HCl buffer (2M, pH 8.0) to the wells and decolorize on a shaker for 10 min to elute the phage antibodies that have strong binding ability to the Enteritidis Salmonella standard; collect the phage eluate in the wells, take 10μL of it and measure its titer after gradient dilution, and the rest is used for amplification.

[0043] The phage eluate was added to fresh E. coli ER2738 bacterial solution and allowed to stand at 37°C for 15 minutes; carbenicillin and SB medium were added, and cultured at 37°C, 220rpm for 2 hours; helper phage M13KO7 (infection multiplicity MOI = 20:1) and kanamycin were added and cultured overnight; the next day, the supernatant was centrifuged and PEG-NaCl solution was added to precipitate and purify the phage. The amplified product was screened for the next round, and the amount of coated antigen was decreased by 10 times. The titer of each round was calculated, and a single clone was picked for amplification and ELISA identification. After 4 rounds of panning, a positive single clone was obtained.

[0044] The above positive monoclonal plasmids were extracted and transformed into E. coli TOP10F' competent cells, and then smeared on solid culture medium for overnight culture after recovery. The next day, a single clone was picked and cultured in LB-carboxybenzyl culture medium, and IPTG was added to induce overnight expression; the next day, the cells were lysed by ultrasonic crusher, filtered by filter membrane, and purified by nickel column to obtain high-purity anti-Salmonella enteritidis nanoantibodies.

[0045] Through amino acid sequencing analysis, the amino acid sequences of the two anti-Salmonella Enteritidis nanobodies obtained are shown in SEQ ID NO: 4 and SEQ ID NO: 9, respectively.

[0046] Amino acid sequence of Nanobody 1 against Salmonella enteritidis (SEQ ID NO: 4) QVQLVESGGGLVQPGGSLRLSRTASRVSLDYYTIAWFRQAPGKEREGVSCISGRGVLSNYADSVKGRFTISRDNAKNTVYLEMSSLEPEDTAVYTCAADAGLRKGTVWYSGASYWGQGTQVTVSS Among them, in Nanobody 1, the amino acid sequence of the complementary determining region CDR1 is: RVSLDYYTIA (SEQ ID NO: 1), the amino acid sequence of the complementary determining region CDR2 is: ISGRGVLSN (SEQ ID NO: 2), and the amino acid sequence of the complementary determining region CDR3 is: AADAGLRKGTVWYSGASY (SEQ ID NO: 3).

[0047] Nucleotide sequence of the gene encoding the anti-Salmonella enteritidis nanobody 1 (SEQ ID NO: 5) CAGGTGCAGCTCGTGGAGTCTGGGGGAGGTTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCAGAACAGCCTCTCGCGTCAGCTTGGATTATTATACCATAGCCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTTTCATGTATTAGTGGTAGAGGGGTCCTGAGCAACTATGCAGACT CCGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGGAAATGAGCAGCCTAGAACCTGAGGACACAGCCGTTTATAACCTGCGCAGCAGATGCGGGCCTCAGAAAGGGTACAGTGTGGTACTCGGGCGCCAGTTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA Amino acid sequence of Nanobody 2 against Salmonella enteritidis (SEQ ID NO: 9) QVQLVESGGGLVQPGGSLRLSRSRPSLDEVTIAWFRQAPGKEREGVSCISGRRVRKNYADSVKGRFTISRDNAKNTVYLEMSSLEPEDTAVYTCARDAISRVGTVWYSFYWGQGTQVTVSS Among them, in Nanobody 2, the amino acid sequence of the complementary determining region CDR1 is: SRPSLDEVTIA (SEQ ID NO: 6), the amino acid sequence of the complementary determining region CDR2 is: ISGRRVRKN (SEQ ID NO: 7), and the amino acid sequence of the complementary determining region CDR3 is: ARDAISRVGTVWYSFY (SEQ ID NO: 8).

[0048] Nucleotide sequence of the gene encoding Nanobody 2 against Salmonella enteritidis (SEQ ID NO: 10) CAGGTGCAGCTCGTGGAGTCTGGGGGAGGTTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCAGATCTCGCCCTAGCTTGGATGAGGTTACCATAGCCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTTTCATGTATTAGTGGTAGACGCGTCAGAAAGAACTATGCAGACT CCGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGGAAATGAGCAGCCTAGAACCTGAGGACACAGCCGTTTATACCTGCGCACGTGATGCGATCTCCAGAGTTGGTACAGTGTGGTACTCGTTCTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA Example 3 Preparation of capture antibody-magnetic bead complex 6×His-Cys (cysteine) was introduced to the C-terminus of the nanobody 1 gene (SEQ ID NO: 5) by PCR amplification technology, and the pET28a-VHH-6×His-Cys recombinant plasmid was constructed and transferred to E. coli BL21(DE3)plysS competent cells. Pick a single clone from the plate and place it in 2YT-Amp medium and culture it overnight; the next day, inoculate 100mL of the culture at a ratio of 1:100 and culture it with shaking until the logarithmic phase; add IPTG to induce at a final concentration of 1mM, 37°C, 220r / min overnight induction; the next day, centrifuge at 8000r / min for 5min to collect the bacterial precipitate, wash the bacterial precipitate twice with PBS phosphate buffer, then take 3-5mL PBS to resuspend the precipitate, use an ultrasonic disruptor to disrupt the E. coli cells in an ice bath environment, and stop the disruption when the bacterial solution is clear and transparent; centrifuge at 8000r / min for 5min to separate the precipitate and supernatant. After the reaction is completed, the reaction solution is placed in a dialysis bag and dialyzed with PBS solution; the solution is changed every 6h, for a total of 5-6 times; the supernatant is collected to obtain the thiolated nanoantibody 1, and the SDS-PAGE image of the thiolated nanoantibody 1 is shown as follows Figure 1 shown.

[0049] Weigh a certain amount of amino magnetic beads (MP, ST412, concentration of 10 mg / mL, purchased from Biyuntian Biotechnology Co., Ltd.), rinse with PBS, discard the supernatant, and repeat this process 3 times; dissolve a certain amount of 3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester crosslinker in dimethylformamide, and mix thoroughly by ultrasonication at 80W for 10 minutes; incubate at room temperature for 1 hour, discard the supernatant, and wash 3 times with PBST; take 3mL of dissolved thiolated nanoantibody 1, resuspend the amino magnetic beads, and mix thoroughly; shake at 220rpm and incubate at room temperature for 1 hour; then, place the capture antibody magnetic bead complex (MP-VHH1) on a magnetic stand, discard the supernatant, and wash with PBST, and repeat this process 3 times; use 3mL of 0.5% gelatin and BSA mixture to fully premix the MP-VHH1 complex, then place it on a magnetic stand and store it at 4°C for use.

[0050] Example 4 Preparation of detection antibodies The nanobody 2 gene (SEQ ID NO: 10) was connected to the alkaline phosphatase gene by PCR amplification technology to obtain the nanobody 2 alkaline phosphatase fusion gene (SEQ ID NO: 12), and the pecan 45-VHH2 recombinant plasmid was constructed; it was transferred into E. coli BL21(DE3)plysS competent cells were plated on 2YT-Amp plates. The next day, single clones were picked, cultured, and transferred to 12L 2YT (Amp, 100μg / ml) liquid culture medium. The bacteria were cultured at 220rpm and 37℃ until the logarithmic phase. The inducer IPTG was added at a final concentration of about 1mmol / L to induce expression overnight. The bacterial precipitate was collected the next day, ultrasonically broken, and Ni was used to generate the precipitate. + Column purification was performed to obtain a detection antibody (Nanobody 2 alkaline phosphatase fusion protein, the amino acid sequence of which is shown in SEQ ID NO: 11). The SDS-PAGE image of the detection antibody is shown in Figure 2 shown.

[0051] Amino acid sequence of fusion protein (SEQ ID NO: 11) QVQLVESGGGLVQPGGSLRLSRSRPSLDEVTIAWFRQAPGKEREGVSCISGRRVRKNYADSVKGRFTISRDNAKNTVYLEMSSLEPEDTAVYTCARDAISRVGTVWYSFYWGQGTQVTVSSEFAAARTPEMPVLENRAAQGDITAPGGARRLTGDQTAALRDSLSDKPAKNIILLIGDGMGDSEITAARNYAEGAGGFFKGIDALPLTGQYTHYALNKKTGKPDYVTDSAASATAWSTGVKTYNGALGVDIHEKDHPTILEMAKAAGLATGNVSTAELQGATPAALVAHVTSRKCYGPSATSEKCPGNALEKGGKGSITEQLLNARADVTLGGGAKTFAETATAGEWQGKTLREQAQARGYQLVSDAASLNSVTEANQQKPLLGLFADGNMPVRWLGPKATYHGNIDKPAVTCTPNPQRNDSVPTLAQMTDKAIELLSKNEKGFFLQVEGASIDKQNHAANPCGQIGETVDLDEAVQRALEFAKKEGNTLVIVTADHAHASQIVAPDTKAPGLTQALNTKDGAVMVMSYGNSEEDSQEHTGSQLRIAAYGPHAANVVGLTDQTDLFYTMKAALGLKVDHHHHHH Nucleotide sequence of the gene encoding the fusion protein (SEQ ID NO: 12) Example 5 Establishment of ELISA kit and detection method for detecting Salmonella enteritidis The ELISA kit comprises a box body, a detachable 96-well ELISA plate arranged in the box body, and reagents arranged in the box body, wherein the reagents comprise the capture antibody magnetic bead complex prepared in Example 3, the detection antibody prepared in Example 4, a Salmonella enteritidis standard solution, a PBS buffer solution, a PBST washing solution, a color developing solution, and a reaction stopping solution.

[0052] The color developing solution is obtained by dissolving 15 mg of p-nitrophenyl phosphate (pNPP) in 15 mL of glycine buffer, and the pH value is 9.6; the reaction termination solution is 3 M sodium hydroxide solution.

[0053] Furthermore, the method for detecting Salmonella enteritidis using the ELISA kit comprises the following steps: 1) The capture antibody magnetic bead complex prepared in Example 3 was coated on a 96-well ELISA plate, with a coating concentration of 100 ng / mL in each well, and reacted at 4°C overnight; the next day, the liquid in the wells was discarded, and the wells were washed three times with PBST containing 0.05% Tween, and the ELISA plate was inverted on absorbent paper and patted dry; 2) Add blocking solution, incubate at 37°C for 30 minutes, shake out the liquid in the wells, wash three times with 0.05% PBST, and turn the ELISA plate upside down on absorbent paper and pat dry; 3) Add 100 μL of standard sample (0 cfu / mL, 10 1 cfu / mL, 10 2 cfu / mL, 10 3 cfu / mL, 10 4 cfu / mL, 10 5 cfu / mL, 10 6 cfu / mL, 10 7 cfu / mL, 10 8 cfu / mL of Enteritidis Salmonella standard solution) or processed samples into each well, repeat 2-4 times for the standard sample and sample, and incubate at 37℃ for 30 minutes; shake out the liquid in the well, wash 3 times with PBST, and turn the ELISA plate upside down on absorbent paper and pat dry; 4) Add 100 μL of the detection antibody prepared in Example 4 at a concentration of 1:2000 (v / v), and incubate at 37° C. for 30 minutes; shake out the liquid in the wells, wash three times with PBST, and invert the ELISA plate on absorbent paper and pat dry; 5) Take the color developing solution, add 100 μL to each well, develop the color for 10-15 minutes in the dark, add 3M sodium hydroxide solution to terminate the reaction, and measure the OD value of each well at a wavelength of 450 nm on an ELISA reader.

[0054] The OD value of each standard well minus the value of the blank well is set as the ordinate, and the corresponding standard concentration is set as the abscissa to draw the standard inhibition curve of Enteritidis Salmonella sample ( Figure 3 ). According to the regression equation of the curve, the concentration of the corresponding sample can be calculated, and the concentration EC of Salmonella enteritidis in the sample can also be calculated. 20 (Lowest detection limit). Through the curve equation (y=y0+a / [1+(x / x0) b ]) can be calculated, EC 20 0.83×10 3 CFU / mL, in the curve equation, y0=0.0833, a=2.5423, b=-0.4679, x0=23090.42.

[0055] In the actual sample detection process, the above method is used to operate and finally the concentration of the enteritidis Salmonella to be detected is calculated.

[0056] The ELISA kit provided by the present invention has the advantage that it can accurately and sensitively detect the content of Salmonella Enteritidis in a sample (piglet feed), overcomes the shortcomings of cumbersome sample pretreatment process, complex operation and time-consuming in instrument detection, and greatly saves detection costs.

[0057] Example 6 Specificity analysis of ELISA method for detection of Salmonella enteritidis The cross-reaction rate is selected to evaluate the specificity of the ELISA method for the Salmonella enteritidis sample, specifically, including the following steps: 1) The capture antibody magnetic bead complex prepared in Example 3 was coated on a 96-well ELISA plate, with a coating concentration of 100 ng / mL in each well, and reacted at 4°C overnight; the next day, the liquid in the wells was discarded, and the wells were washed three times with PBST containing 0.05% Tween, and the ELISA plate was inverted on absorbent paper and patted dry; 2) Add blocking solution, incubate at 37°C for 30 minutes, shake out the liquid in the wells, wash three times with 0.05% PBST, and turn the ELISA plate upside down on absorbent paper and pat dry; 3) Prepare the concentrations of 0 cfu / mL and 10 1 cfu / mL, 10 2 cfu / mL, 10 3 cfu / mL, 10 4 cfu / mL, 10 5 cfu / mL, 10 6 cfu / mL, 10 7 cfu / mL, 10 8cfu / mL of Enteritidis Salmonella sample, Salmonella typhimurium solution, and Salmonella choleraesuis solution, take 100 μL of the above bacterial solution and add it to each well, repeat 2 to 4 times; incubate at 37°C for 30 minutes; shake out the liquid in the well, wash 3 times with PBST, and turn the ELISA plate upside down on absorbent paper to pat dry; 4) Add 100 μL of the detection antibody prepared in Example 4 at a concentration of 1:2000 (v / v), and incubate at 37° C. for 30 minutes; shake out the liquid in the wells, wash three times with PBST, and invert the ELISA plate on absorbent paper and pat dry; 5) Take the color developing solution and add 150 μL to each well. Protect from light and color for 15-20 minutes. Add 3M sodium hydroxide solution to terminate the reaction. Measure the OD value of each well at a wavelength of 405 nm on an ELISA reader.

[0058] Calculate the EC of each test bacterial solution 50 Value, using the formula, cross-reaction rate = [EC 50 (Salmonella Enteritidis sample) / EC 50 (other bacteria)] × 100%, the cross-reaction rate can be calculated and the results are shown in Table 4 below.

[0059] Table 4 Cross-reaction rate results

[0060] As can be seen from Table 4, the cross-reaction rate is less than 0.5%, indicating that the ELISA analysis method has good specificity for Salmonella Enteritidis samples.

[0061] In summary, the kit provided by the present invention has the advantages of good specificity and high sensitivity when detecting the content of Salmonella Enteritidis in a sample.

[0062] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0063] The above-mentioned embodiments only express the 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, which 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. A nanobody against Salmonella enteritidis, characterized in that: The Nanobody comprises at least one of the following: A1) a Nanobody comprising a complementary determining region CDR1 as shown in SEQ ID NO: 1, a complementary determining region CDR2 as shown in SEQ ID NO: 2, and a complementary determining region CDR3 as shown in SEQ ID NO: 3; A2) A Nanobody comprising a complementary determining region CDR1 as shown in SEQ ID NO: 6, a complementary determining region CDR2 as shown in SEQ ID NO: 7, and a complementary determining region CDR3 as shown in SEQ ID NO: 8, respectively.

2. The Nanobody according to claim 1, characterized in that The amino acid sequence of the Nanobody is selected from any one of the following: B1) having the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 9; B2) An amino acid sequence having the same function as the amino acid sequence defined in B1) by substitution, deletion or addition of one or more amino acids; B3) an amino acid sequence having a sequence identity of more than 80% and the same function as the amino acid sequence defined in B1) or B2); B4) The amino acid sequence obtained by connecting a tag to the N-terminus and / or C-terminus of B1).

3. A nucleic acid molecule encoding a Nanobody according to any one of claims 1 to 2.

4. The nucleic acid molecule according to claim 3, characterized in that The nucleic acid molecule is selected from any of the following nucleic acid molecules: C1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 5 or SEQ ID NO: 10; C2) a nucleic acid molecule that hybridizes with the nucleic acid molecule defined in C1) and encodes the Nanobody according to any one of claims 1 to 2; C3) A nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in C1) or C2) and encodes the Nanobody according to any one of claims 1 to 2.

5. A recombinant vector, characterized in that: Comprising the nucleic acid molecule according to any one of claims 3-4.

6. A recombinant cell, characterized in that Comprising the nucleic acid molecule according to any one of claims 3-4 or the recombinant vector according to claim 5.

7. A method for preparing a nanobody against Salmonella enteritidis, characterized in that: The steps include: Cultivating the recombinant cell according to claim 6, inducing expression, and obtaining a culture; The Nanobody of any one of claims 1-2 is isolated from said culture.

8. A kit for detecting Salmonella enteritidis, characterized in that: including a capture antibody magnetic bead complex and / or a detection antibody; Wherein, the capture antibody-magnetic bead complex is obtained by compounding the nano-antibody described in claim 1 A1) with magnetic beads, and the magnetic beads are ferroferric oxide particles having primary amino groups on the surface, and the particle size of the ferroferric oxide particles is 180-220 nm; The detection antibody is a fusion protein obtained by fusing the nanobody described in A2) of claim 1 with alkaline phosphatase, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:

11.

9. The kit according to claim 8, characterized in that The method also comprises at least one of a standard solution of Salmonella enteritidis, a buffer solution, a washing solution, a color developing solution and a reaction stopping solution.

10. Use of the Nanobody according to any one of claims 1-2, the nucleic acid molecule according to any one of claims 3-4, the recombinant vector according to claim 5, the recombinant cell according to claim 6, the Nanobody prepared by the preparation method according to claim 7, and the kit according to any one of claims 8-9 in the detection of Enteritidis Salmonella and / or in the preparation of a product for detecting Enteritidis Salmonella.

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