A nanobody against salmonella enteritidis, a kit and use thereof

Abstract

The application discloses an anti-Salmonella enteritidis nanobody, a kit and application thereof, and belongs to the technical field of genetic engineering. The nanobody comprises at least one of the following: A1) a nanobody comprising a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, wherein the amino acid sequences of the complementarity determining region CDR1, the complementarity determining region CDR2 and the complementarity determining region CDR3 are respectively shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; and A2) a nanobody comprising a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, wherein the amino acid sequences of the complementarity determining region CDR1, the complementarity determining region CDR2 and the complementarity determining region CDR3 are respectively shown as SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. The anti-Salmonella enteritidis nanobody provided by the application has high affinity and good specificity. Therefore, the anti-Salmonella enteritidis nanobody has a good application prospect in the detection of Salmonella enteritidis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genetic engineering, and particularly relates to a nano antibody against Salmonella enteritidis, a kit and application thereof. BACKGROUND

[0002] Salmonella enteritidis is one of the main pathogens causing salmonellosis in piglets, and is more common in weaned piglets. The prevalence of salmonellosis in piglets is affected by various factors, including the age, health status, feeding management, seasonal variation and regional differences of the pig population. Feed is one of the important transmission media of salmonellosis in piglets, and the presence of Salmonella enteritidis in pig feed is highly hazardous 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 pig feed, strict biosecurity 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. The conventional detection methods for Salmonella enteritidis include pre-enrichment, selective enrichment, plate separation, biochemical test, serological identification, polymerase chain reaction (PCR), real-time fluorescent quantitative PCR, etc. Although these analysis methods have high accuracy, they require expensive instruments, and the sample separation, extraction, purification, derivatization and other pretreatments are complex, the analysis speed is slow, the detection sensitivity is low, and it is difficult to realize on-site rapid detection. With the rapid increase in the amount of samples to be tested, especially the requirement for on-site rapid detection, traditional analysis methods are difficult to meet the requirements, and therefore, there is an urgent need to develop an efficient and convenient detection method for Salmonella enteritidis.

[0003] Immune magnetic 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 on-site rapid detection, and can provide an efficient and convenient detection method for the detection of Salmonella enteritidis residues in feed. The selection of antibodies is the core of the immune analysis method. Compared with conventional antibodies, nano antibodies (VHH) have the characteristics of small molecular weight, easy expression and low production cost, and nano antibodies have the advantages of relatively simple preparation process and low antibody cross-reaction.

[0004] However, the existing nano antibodies for detecting Salmonella enteritidis still have problems such as low sensitivity and poor specificity. SUMMARY

[0005] The purpose of the present application is to provide a nano antibody against Salmonella enteritidis, a kit and application thereof. To solve the problem that the existing nano antibodies for detecting Salmonella enteritidis still have low sensitivity and poor specificity.

[0006] In a first aspect, the present application provides a nanobody against Salmonella enterica, the nanobody comprising at least one of: A1 ) a nanobody comprising a complementarity determining region CDR1 as shown in SEQ ID NO: 1, a complementarity determining region CDR2 as shown in SEQ ID NO: 2, a complementarity determining region CDR3 as shown in SEQ ID NO: 3; A2) a nanobody comprising a complementarity determining region CDR1 as shown in SEQ ID NO: 6, a complementarity determining region CDR2 as shown in SEQ ID NO: 7, a complementarity determining region CDR3 as shown in SEQ ID NO: 8.

[0007] The nanobody provided by the present application (also referred to as single domain antibody) has only the variable region (VHH) of the heavy chain antibody, which in turn consists of a framework region FR1, a complementarity determining region CDR1, a framework region FR2, a complementarity determining region CDR2, a framework region FR3, a complementarity determining region CDR3 and a framework region FR4.

[0008] In the present application, the term "nanobody" generally refers to an antibody consisting of only one H chain V region, which can also be referred to as VHH antibody. The ability of the nanobody to bind to an antigen and its stability are essentially identical to those of a complete antibody. In the present text, unless the context clearly indicates otherwise, when the term "nanobody" is mentioned, it not only includes the complete nanobody, but also antigen-binding fragments of the nanobody. The antigen-binding fragment refers to a polypeptide comprising a fragment of the nanobody, which 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 "antigen-binding portion".

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

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

[0011] In a second aspect, the present application provides a nucleic acid molecule encoding any of the above-mentioned nanobodies.

[0012] The above-mentioned nucleic acid molecules provided by the present application can generally 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 set forth in SEQ ID NO: 5 or SEQ ID NO: 10; C2) a nucleic acid molecule hybridizing under stringent conditions to the nucleic acid molecule defined in C1) and encoding any of the above-mentioned nanobodies; C3) a nucleic acid molecule having more than 90% sequence identity to the nucleic acid molecule defined in C1) or C2) and encoding any of the above-mentioned nanobodies.

[0014] As used herein, the term "hybridizes under stringent conditions" means that two nucleic acid molecule fragments hybridize to each other under standard hybridization conditions as described in the section "Expression of cloned genes in E. coli" of Sambrook et al., Molecular Cloning: A Laboratory Manual (1989) (Cold Spring Harbor Laboratory Press, New York, USA). Such conditions are, for example, hybridization in 6.0x SSC at 45°C followed by a washing step in 2x SSC at 50°C. To select the stringency, the salt concentration in the washing step can be chosen, for example, between 2.0x SSC at 50°C for low stringency and 2.0x SSC at 50°C for high stringency. In addition, the temperature in the washing step can vary between about 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 eye or by computer software, such as the software programs described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. When a position in compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position. A degree of identity between two or more sequences can be expressed as a percentage (%) which can be used to evaluate the identity between related sequences. A polynucleotide sequence or an amino acid sequence has a certain percentage (e.g. 90%, 95%, 98% or 99%) of "sequence identity" to another sequence if the percentage of bases or amino acids in the two sequences that are identical when the sequences are aligned

[0016] In a third aspect, the present application provides a recombinant vector comprising any one of the nucleic acid molecules described above.

[0017] The recombinant vector in the present application includes a cloning vector for replicating relevant sequences and an expression vector for expressing relevant genes. For example, the expression vector in the present application can be pET28a.

[0018] In a fourth aspect, the present application provides a recombinant cell comprising any one of the nucleic acid molecules described above or the recombinant vector described above.

[0019] In a fifth aspect, the present application provides a method for preparing a nanobody against Salmonella enteritidis, comprising the following steps: culturing the recombinant cell described above, inducing expression to obtain a culture; and isolating any one of the nanobodies described above from the culture.

[0020] In the present application, the culture method and culture conditions have no special requirements, as long as the normal growth of the recombinant cell is ensured. And the method for isolating the nanobody described above from the culture is a conventional method in the art.

[0021] In a sixth aspect, the present application provides a kit for detecting Salmonella enteritidis, comprising a capture antibody magnetic bead complex and / or a detection antibody; wherein the capture antibody magnetic bead complex is obtained by complexing the nanobody A1) described above 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; and the detection antibody is a fusion protein obtained by fusing the nanobody A2) described above with alkaline phosphatase, and the amino acid sequence of the fusion protein is shown as SEQ ID NO: 11.

[0022] In some embodiments, the concentration of the capture antibody magnetic bead complex is 80-120 ng / mL, preferably 100 ng / mL; and the concentration of the detection antibody is 0.1-0.5 μg / mL, preferably 0.3 μg / mL.

[0023] 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 termination solution.

[0024] In some preferred embodiments, the buffer solution is a PBS buffer solution; the washing solution is a PBST washing solution; the color developing solution is obtained by dissolving 15 mg of p-nitrophenyl phosphate (pNPP) in 15 mL of glycine buffer solution, and has a pH value of 9.6; and the reaction termination solution is a 3M sodium hydroxide solution.

[0025] It can be understood that the kit further comprises a box body, and an enzyme-labeled plate detachably arranged in the box body.

[0026] In a seventh aspect, the present application provides use of any one of the nanobodies, any one of the nucleic acid molecules, the recombinant vector, the recombinant cell, the nanobodies prepared by the preparation method, or any one of the kits in detection of Salmonella enteritidis and / or for preparing a product for detecting Salmonella enteritidis.

[0027] In the present application, when performing actual sample analysis and detection, the capture antibody magnetic bead complex and the Salmonella enteritidis sample to be detected are sequentially added to each well of the closed enzyme-labeled plate, and placed on a 96-well magnetic stand; the nanobodies on the magnetic beads and the Salmonella enteritidis to be detected interact and bind; then the detection antibody (a fusion protein obtained by fusing the nanobodies with alkaline phosphatase) and the Salmonella enteritidis are added and combined; since the content of the magnetic beads and the detection antibody in each well is consistent, when the concentration of the Salmonella enteritidis to be detected is high, the Salmonella enteritidis is combined with the coating antibody and the detection antibody more; finally, the color developing solution and the reaction termination solution are added; the color developing reaction is deep, the OD value detected by the enzyme-labeled instrument is high, indicating that the content of Salmonella enteritidis in the sample is high; on the contrary, when the concentration of the Salmonella enteritidis to be detected is low, the measured OD value is low, indicating that the content of Salmonella enteritidis in the sample is low. According to the standard curve drawn by detecting the Salmonella enteritidis standard solution with a known concentration, the concentration of the Salmonella enteritidis to be detected can be calculated.

[0028] The present application has the following beneficial effects: Different from the prior art, the nanobodies against Salmonella enteritidis provided by the present application have strong affinity and good specificity; when the kit containing the nanobodies is used to detect the content of Salmonella enteritidis in a sample, the kit has the advantages of good specificity and high sensitivity, and can quickly and in large quantities detect samples, and therefore has good application prospects in detection of Salmonella enteritidis. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 SDS-PAGE of the thiolated nanobody 1 prepared in Example 3 of the present application, wherein lane M: marker, lane 1: thiolated nanobody 1;

[0030] Figure 2 SDS-PAGE of the detection antibody (nanobody 2 alkaline phosphatase fusion protein) prepared in Example 4 of the present application, wherein lane M: marker, lane 1: nanobody 2 alkaline phosphatase fusion protein;

[0031] Figure 3 Standard curve of Salmonella enteritidis in Example 5 of the present application. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present application.

[0033] The experimental methods not specified in the embodiments are generally carried out according to the 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 M. R. Green, Molecular Biology by Robert·F·Weaver, or the experimental methods suggested by the manufacturers of reagent kits and instrument equipment. The reagents and biological materials used in the embodiments can be obtained from commercial channels if not otherwise specified.

[0034] Example 1 Construction of phage display nanobody library of Salmonella enteritidis

[0035] The Salmonella enteritidis (10 9 The Salmonella enteritidis (10 SfiI The pComb3X plasmid vector and the scFv gene fragment are subjected to enzyme cutting, the VHH gene fragment is connected to the phagemid pComb3x through T4 ligase, and high-efficiency electroporation is performed on Escherichia coli ER2738 to construct a Salmonella enteritidis phage nanobody library. It is determined that the primary library capacity reaches 10 8 cfu / mL, the helper phage (multiplicity of infection is 20:1) M13KO7 is added for rescue, and a phage nanobody library (phage display VHH library) is obtained, and the library capacity is 10 13 pfu / mL, and the library diversity is good.

[0036] In the reverse transcription PCR, the reverse transcription kit is M-MLV first-strand cDNA synthesis kit (purchased from OMEGA company), and the reverse transcription system is shown in Table 1.

[0037] Table 1 Reverse transcription system

[0038]

[0039] The reagent is prepared according to the reverse transcription system in Table 1, and reverse transcription is performed at 42°C for 30 min to obtain cDNA.

[0040] The cDNA obtained above is subjected to two rounds of overlapping PCR amplification, wherein the primer sequences (5'-3') of PCR amplification are as follows:

[0041] GSP-RT: CGCCATCAATRTACCAGTTGA (SEQ ID NO: 13);

[0042] LP-leader: GTGGTCCTGGCTGCTCTW (SEQ ID NO: 14);

[0043] F: CATGCCATGACTGTGGCCCAGGCGGCCCAGKTGCAGCTCGTGGAGTC (SEQ ID NO: 15);

[0044] R: CATGCCATGACTCGCGGCCGGCCTGGCCATGGGGGTCTTCGCTGTGGTGCG (SEQ ID NO: 16);

[0045] Wherein, R represents base A / G, W represents base A / T, and K represents base G / T.

[0046] The reaction system of the first round of overlapping PCR is shown in Table 2.

[0047] Table 2 Reaction system

[0048]

[0049] The reaction procedure of the first round of overlap PCR is as follows:

[0050] 95°C pre-denaturation for 3 min, 94°C for 30 s, 57°C for 30 s, 72°C for 45 s (25 cycles), 72°C for 10 min, 4°C for ∞.

[0051] The reaction system of the second round of overlap PCR is shown in Table 3.

[0052] Table 3 Reaction system

[0053]

[0054] The reaction procedure of the second round of overlap PCR is as follows:

[0055] 95°C pre-denaturation for 3 min, 94°C for 30 s, 57°C for 30 s, 72°C for 45 s (30 cycles), 72°C for 10 min, 4°C for ∞.

[0056] Example 2 Screening of Salmonella enteritidis-specific nanobodies

[0057] The first well of a 96-well enzyme-labeled plate was coated with Salmonella enteritidis at a concentration of 10 5 cfu / mL and was left overnight at 4°C; the next day, the coating solution was poured out, and the wells were washed with PBST three times, 100 μL of 1% gelatin was added to the first two wells, 100 μL of 1% BSA solution was added to the last four wells, and the wells were incubated at room temperature for 1 h; 110 μL of the phage-displayed VHH library obtained in Example 1 was added to 110 μL of 3% BSA solution, and the mixture was shaken at 220 rpm at 25°C for 1 h to allow the phage to bind to the BSA and remove non-specific phage antibodies; the solution was discarded, and the wells were washed with PBST three times, each time for 1 min; 100 μL of the premixed library was added to the first two wells, and the wells were shaken at room temperature for 2 h to allow the phage to bind to the coating; the solution was discarded, and the wells were washed with PBST three times, each time for 1 min; 100 μL of Gly-HCl buffer (0.2 M, pH 2.2) was added, and the wells were shaken for 10 min; then, 20 μL of Tris-HCl buffer (2 M, pH 8.0) was added, and the wells were shaken for 10 min to elute the phage antibodies that had a strong binding capacity to the Salmonella enteritidis standard; the phage eluate in the wells was collected, 10 μL of which was diluted and used to determine the titer, and the rest was used for amplification.

[0058] The phage eluent was added to fresh E. coli ER2738 bacterial solution, and was allowed to stand at 37°C for 15 min; carbenicillin and SB medium were added, and the solution was cultured at 37°C, 220 rpm, for 2 h; helper phage M13KO7 (multiplicity of infection MOI = 20:1) and kanamycin were added, and the solution was cultured overnight; the next day, the supernatant was obtained by centrifugation, and PEG-NaCl solution was added to precipitate and purify the phage. The amplified product was subjected to the next round of screening, the amount of coated antigen was reduced by 10 times, the titer of each round was calculated, and a single clone was picked and amplified and subjected to ELISA identification. After 4 rounds of screening, a positive single clone was obtained.

[0059] The plasmid of the positive single clone was extracted, and was transformed into E. coli TOP10F' competent cells, respectively. After recovery, the cells were plated on solid medium and were cultured overnight. The next day, a single clone was picked and was cultured in LB-carbenicillin medium, IPTG was added to induce expression overnight; the next day, the cells were lysed by an ultrasonic disrupter, were filtered by a filter membrane, and were purified by a nickel column to obtain high-purity anti-Salmonella enteritidis nanobodies.

[0060] According to 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.

[0061] Amino acid sequence of anti-Salmonella enteritidis nanobody 1 (SEQ ID NO: 4)

[0062] QVQLVESGGGLVQPGGSLRLSRTASRVSLDYYTIAWFRQAPGKEREGVSCISGRGVLSNYADSVKGRFTISRDNAKNTVYLEMSSLEPEDTAVYTCAADAGLRKGTVWYSGASYWGQGTQVTVSS

[0063] In the nanobody 1, the amino acid sequence of the complementarity determining region CDR1 is RVSLDYYTIA (SEQ ID NO: 1), the amino acid sequence of the complementarity determining region CDR2 is ISGRGVLSN (SEQ ID NO: 2), and the amino acid sequence of the complementarity determining region CDR3 is AADAGLRKGTVWYSGASY (SEQ ID NO: 3).

[0064] Nucleotide sequence of the gene encoding anti-Salmonella enteritidis nanobody 1 (SEQ ID NO: 5)

[0065] CAGGTGCAGCTCGTGGAGTCTGGGGGAGGTTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCAGAACAGCCTCTCGCGTCAGCTTGGATTATTATACCATAGCCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTTTCATGTATTAGTGGTAGAGGGGTCCTGAGCAACTATGCAGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGGAAATGAGCAGCCTAGAACCTGAGGACACAGCCGTTTATACCTGCGCAGCAGATGCGGGCCTCAGAAAGGGTACAGTGTGGTACTCGGGCGCCAGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0066] Amino acid sequence of Nanobody 2 against Salmonella enteritidis (SEQ ID NO: 9)

[0067] QVQLVESGGGLVQPGGSLRLSRSRPSLDEVTIAWFRQAPGKEREGVSCISGRRVRKNYADSVKGRFTISRDNAKNTVYLEMSSLEPEDTAVYTCARDAISRVGTVWYSFYWGQGTQVTVSS

[0068] In Nanobody 2, the amino acid sequence of the complementarity determining region CDR1 is SRPSLDEVTIA (SEQ ID NO: 6), the amino acid sequence of the complementarity determining region CDR2 is ISGRRVRKN (SEQ ID NO: 7), and the amino acid sequence of the complementarity determining region CDR3 is ARDAISRVGTVWYSFY (SEQ ID NO: 8).

[0069] Nucleotide sequence of the gene encoding Nanobody 2 against Salmonella enteritidis (SEQ ID NO: 10)

[0070] CAGGTGCAGCTCGTGGAGTCTGGGGGAGGTTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCAGATCTCGCCCTAGCTTGGATGAGGTTACCATAGCCTGGTTCCGCCAGGCCCCAGGGAAGGAGCGTGAGGGGGTTTCATGTATTAGTGGTAGACGCGTCAGAAAGAACTATGCAGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGGAAATGAGCAGCCTAGAACCTGAGGACACAGCCGTTTATACCTGCGCACGTGATGCGATCTCCAGAGTTGGTACAGTGTGGTACTCGTTCTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0071] Example 3 Preparation of the capture antibody magnetic bead complex

[0072] The 6xHis-Cys (Cysteine) was introduced to the C-terminal of the Nanobody 1 gene (SEQ ID NO: 5) by PCR amplification technology, and the pET28a-VHH-6xHis-Cys recombinant plasmid was constructed and transferred to E. coli BL21(DE3)plysS competent cells. Single colonies were picked from the plate and cultured in 2YT-Amp medium overnight; the next day, 100 mL of culture was inoculated at 1:100 and incubated at 220 r / min until the logarithmic phase; IPTG was added to induce a final concentration of 1 mM, and the culture was induced at 37°C overnight; the next day, the bacterial pellet was collected by centrifugation at 8000 r / min for 5 min, and the bacterial pellet was washed twice with PBS phosphate buffer; 3-5 mL of PBS was used to resuspend the pellet, and the E. coli cells were broken by ultrasonic disrupter in an ice bath environment until the bacterial solution was clear and transparent; the bacteria were broken by centrifugation at 8000 r / min for 5 min. After the reaction was completed, the reaction solution was placed in a dialysis bag and dialyzed with PBS; the solution was changed every 6 h, for a total of 5-6 times; the supernatant was collected, and the thiolated Nanobody 1 was obtained. The SDS-PAGE diagram of the thiolated Nanobody 1 is shown in Figure 1 .

[0073] A certain amount of amino magnetic beads (MP, ST412, concentration of 10 mg / mL, purchased from Biyun Tian Biotechnology Co., Ltd.) was washed with PBS, and the supernatant was discarded. The operation was repeated for 3 times. A certain amount of 3-(2-pyridyl disulfide) propionic acid N-hydroxy succinimidyl ester crosslinking agent was dissolved in dimethylformamide, and 80W ultrasonic was used for 10 min to make it fully mixed. Incubation was performed at room temperature for 1 h, the supernatant was discarded, and PBST was used for washing 3 times. 3 mL of dissolved thiolated nanobody 1 was taken, the amino magnetic beads were resuspended, and they were fully mixed. Incubation was performed at room temperature for 1 h on a shaking bed at 220 rpm. Then, the capture antibody magnetic bead complex (MP-VHH1) was rested on a magnetic stand, the supernatant was discarded, and PBST was used for washing, which was repeated for 3 times. 3 mL of 0.5% gelatin and BSA mixture was used, and the MP-VHH1 complex was fully premixed, and then placed on a magnetic stand and stored at 4°C for later use.

[0074] Example 4 Preparation of detection antibody

[0075] The nanobody 2 gene (SEQ ID NO: 10) was connected with the alkaline phosphatase gene by PCR amplification technology to obtain a nanobody 2 alkaline phosphatase fusion gene (SEQ ID NO: 12), and a pecan 45-VHH2 recombinant plasmid was constructed. The recombinant plasmid was transformed into E. coli BL21 (DE3) plysS competent cells, and then coated on a 2YT-Amp plate. The next day, a single colony was picked, cultured, and transferred to a 1 2L 2YT (Amp, 100 μg / ml) liquid medium. The bacteria were cultured at 220 rpm and 37°C until the logarithmic phase, and then an inducer IPTG with a final concentration of about 1 mmol / L was added for overnight induction expression. The next day, the bacterial precipitate was collected, ultrasonically broken, and purified by Ni + column to obtain a detection antibody (nanobody 2 alkaline phosphatase fusion protein, amino acid sequence as shown in SEQ ID NO: 11). The SDS-PAGE diagram of the detection antibody is shown in Figure 2

[0076] Amino acid sequence of the fusion protein (SEQ ID NO: 11)

[0077] ​QVQLVESGGGLVQPGGSLRLSRSRPSLDEVTIAWFRQAPGKEREGVSCISGRRVRKNYADSVKGRFTISRDNAKNTVYLEMSSLEPEDTAVYTCARDAISRVGTVWYSFYWGQGTQVTVSSEFAAARTPEMPVLENRAAQGDITAPGGARRLTGDQTAALRDSLSDKPAKNIILLIGDGMGDSEITAARNYAEGAGGFFKGIDALPLTGQYTHYALNKKTGKPDYVTDSAASATAWSTGVKTYNGALGVDIHEKDHPTILEMAKAAGLATGNVSTAELQGATPAALVAHVTSRKCYGPSATSEKCPGNALEKGGKGSITEQLLNARADVTLGGGAKTFAETATAGEWQGKTLREQAQARGYQLVSDAASLNSVTEANQQKPLLGLFADGNMPVRWLGPKATYHGNIDKPAVTCTPNPQRNDSVPTLAQMTDKAIELLSKNEKGFFLQVEGASIDKQNHAANPCGQIGETVDLDEAVQRALEFAKKEGNTLVIVTADHAHASQIVAPDTKAPGLTQALNTKDGAVMVMSYGNSEEDSQEHTGSQLRIAAYGPHAANVVGLTDQTDLFYTMKAALGLKVDHHHHHH

[0078] Nucleotide sequence of the gene encoding the fusion protein (SEQ ID NO: 12)

[0079]

[0080] Example 5: Establishment of ELISA kit and detection method for detecting Salmonella enteritidis

[0081] The ELISA kit comprises a box body, a detachable 96-well enzyme-labeled 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, a PBST washing solution, a color developing solution, and a reaction termination solution.

[0082] The color developing solution is obtained by dissolving 15 mg of p-nitrophenyl phosphate (pNPP) in 15 mL of glycine buffer, and has a pH value of 9.6; and the reaction termination solution is a 3M sodium hydroxide solution.

[0083] Further, the method for detecting Salmonella enteritidis by using the ELISA kit comprises the following steps:

[0084] 1) The capture antibody magnetic bead complex prepared in Example 3 is coated on the 96-well enzyme-labeled plate, and the coating concentration of each well is 100 ng / mL, and the reaction is carried out at 4°C overnight; the next day, the liquid in the well is shaken off, and the plate is washed with 0.05% PBST for 3 times, and then the plate is inverted on the absorbent paper and dried;

[0085] 2) The blocking solution is added, and the plate is incubated at 37°C for 30 minutes; the liquid in the well is shaken off, and the plate is washed with 0.05% PBST for 3 times, and then the plate is inverted on the absorbent paper and dried;

[0086] 3) 100 μL of the 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 the Salmonella enteritidis standard solution) or the treated sample is added to each well, and the standard sample and the sample are repeated for 2-4 times, and the plate is incubated at 37°C for 30 minutes; the liquid in the well is shaken off, and the plate is washed with PBST for 3 times, and then the plate is inverted on the absorbent paper and dried;

[0087] 4) 100 μL of the detection antibody prepared in Example 4 is added at a ratio of 1:2000 (v / v), and the plate is incubated at 37°C for 30 minutes; the liquid in the well is shaken off, and the plate is washed with PBST for 3 times, and then the plate is inverted on the absorbent paper and dried;

[0088] 5) Take the color developing solution, add 100 μL to each well, develop color in dark for 10-15 minutes, 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 enzyme label instrument.

[0089] Subtract the value of the blank hole from the OD value of each standard hole to obtain the vertical coordinate, and the corresponding standard concentration is the horizontal coordinate, to draw the standard inhibition curve of the Salmonella enteritidis sample Figure 3 ). According to the regression equation of the curve, the concentration of the corresponding sample can be obtained, and the concentration EC 20 (the minimum detection limit) of the Salmonella enteritidis sample can also be obtained. The EC b can be calculated through the curve equation (y=y0+a / [1+(x / x0) 20 ). The EC 3 is 0.83x10 1 CFU / mL, and in the curve equation, y0=0.0833, a=2.5423, b=-0.4679, and x0=23090.42.

[0090] In the actual sample detection process, the concentration of the Salmonella enteritidis to be detected is calculated through the above method.

[0091] The ELISA kit provided by the application has the advantages that the content of the Salmonella enteritidis in the sample (piglet feed) can be accurately and sensitively detected, the shortcomings of a complicated and time-consuming sample pretreatment process, complex operation and the like in instrument detection are overcome, and the detection cost is greatly saved.

[0092] Example 6: Specific analysis of the ELISA method for Salmonella enteritidis detection

[0093] The cross reactivity is selected to evaluate the specificity of the ELISA method for the Salmonella enteritidis sample, and specifically, the following steps are included:

[0094] 1) The capture antibody magnetic bead complex prepared in Example 3 is coated on a 96-well enzyme label plate, and the coating concentration of each well is 100 ng / mL, and the reaction is carried out at 4°C overnight; the next day, the liquid in the well is shaken off, washed with PBST containing 0.05% Tween for 3 times, and the enzyme label plate is inverted on the absorbent paper and dried;

[0095] 2) Add blocking solution, incubate at 37°C for 30 minutes, shake off the liquid in the well, wash with 0.05% PBST for 3 times, and invert the enzyme label plate on the absorbent paper and dry;

[0096] 3) Prepare 0 cfu / mL, 10 1 cfu / mL, 10 2 cfu / mL, 10 3 cfu / mL, 10 4 cfu / mL, 105 cfu / mL, 10 6 cfu / mL, 10 7 cfu / mL, 10 8 cfu / mL of Salmonella enteritidis sample, Salmonella typhimurium liquid, Salmonella choleraesuis liquid, 100 muL of the above-mentioned bacterial liquid was added to each well, and 2-4 repeats were carried out; 37 DEG C incubation for 30 minutes; the liquid in the well was shaken out, washed with PBST for 3 times, and the enzyme-labeled plate was inverted on the absorbent paper and dried;

[0097] 4) 100 muL of the detection antibody prepared in Example 4 was added 1:2000 (v / v), 37 DEG C incubation for 30 minutes; the liquid in the well was shaken out, washed with PBST for 3 times, and the enzyme-labeled plate was inverted on the absorbent paper and dried;

[0098] 5) Take the color developing liquid, add 150 muL per well, develop color for 15-20 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 405nm on the enzyme-labeled instrument.

[0099] The EC 50 value of each kind of to-be-tested bacterial liquid was calculated, and the cross reaction rate was calculated by the formula: cross reaction rate = [EC 50 (Salmonella enteritidis sample) / EC 50 (other bacteria)] x 100%, and the results are shown in the following table 4.

[0100] Table 4 Cross reaction rate results

[0101]

[0102] As can be seen from table 4, the cross reaction rate is less than 0.5%, which indicates that the ELISA analysis method has good specificity for Salmonella enteritidis sample.

[0103] In summary, when the kit provided by the application is used to detect the content of Salmonella enteritidis in a sample, the kit has the advantages of good specificity and high sensitivity.

[0104] It should be noted that each of the above embodiments belongs to the same inventive concept, and the description of each embodiment has its own emphasis. If not fully described in an individual embodiment, reference can be made to the description in other embodiments.

[0105] The above-described embodiments only express the implementation of the application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are within the scope of protection of the application. Therefore, the protection scope of the patent of the application should be subject to the appended claims.

Claims

1. A nanobody composition for combating Salmonella enteritidis, characterized in that, The nanobody composition comprises: A1) A nanobody containing the amino acid sequences of complementarity-determining region CDR1 as shown in SEQ ID NO: 1, complementarity-determining region CDR2 as shown in SEQ ID NO: 2, and complementarity-determining region CDR3 as shown in SEQ ID NO: 3; and A2) A nanobody containing complementarity-determining regions CDR1 as shown in SEQ ID NO: 6, CDR2 as shown in SEQ ID NO: 7, and CDR3 as shown in SEQ ID NO: 8, respectively.

2. The nanobody composition according to claim 1, characterized in that, The amino acid sequence of the nanobody composition is shown in SEQ ID NO: 4 or SEQ ID NO:

9.

3. The nucleic acid molecule encoding the nanobody composition according to any one of claims 1-2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 5 or SEQ ID NO:

10.

5. A recombinant vector, characterized in that, It comprises any one of the nucleic acid molecules according to claims 3-4.

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

7. A method for preparing a nanobody composition against Salmonella enteritidis, characterized in that, Includes the following steps: The recombinant cells of claim 6 were cultured and induced to express the desired expression to obtain a culture. Isolate the nanobody composition according to any one of claims 1-2 from the culture.

8. A kit for detecting Salmonella enteritidis, characterized in that, This includes capturing antibody-magnetic bead complexes and / or detecting antibodies; The captured antibody magnetic bead complex is obtained by combining the nanoantibody described in claim 1 (A1) with magnetic beads, wherein the magnetic beads are iron oxide particles with primary amino groups on their surface, and the particle size of the iron oxide particles is 180-220 nm. The detection antibody is a fusion protein obtained by fusing the nanobody described in claim 1 (A2) with alkaline phosphatase, and the amino acid sequence of the fusion protein is shown in SEQ ID NO:

11.

9. The reagent kit according to claim 8, characterized in that, It also includes at least one of the following: Salmonella enteritidis standard solution, buffer solution, washing solution, colorimetric solution, and reaction termination solution.

10. The use of the nanobody composition 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 composition prepared by the preparation method according to claim 7, and the kit according to any one of claims 8-9 in the preparation of products for detecting Salmonella enteritidis.

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