CN106008711A - Vomiting toxin antigen-antibody immune complex-specific binding variable domain of heavy chain of heavy chain antibody and application thereof - Google Patents

Abstract

The invention belongs to the field of biotechnology, in particular to the preparation and application of a single domain heavy chain antibody (Variable domain of heavy chain of heavy chain antibody, VHH) that can specifically bind to the vomitoxin antigen-antibody immune complex (Immunocomplex), and its amino acid sequence SEQ ID NO.1. The single-domain heavy chain antibody of the invention can specifically bind to the vomitoxin antigen-antibody immune complex, can replace traditional antibodies, and can be applied to non-competitive immunological analysis of DON. The amino acid sequence provided by the present invention can be used as a precursor to be modified by random or site-directed mutagenesis techniques to obtain mutants with better properties, which can be used to develop proteins or polypeptides that are further used in the fields of industry and food safety.

Description

Single domain heavy chain antibody specifically binding to vomitoxin antigen-antibody immune complex and application thereof

technical field

The present invention relates to single-domain heavy chain antibody technology (also known as nanobody technology) and genetic engineering antibody technology, belonging to the field of biotechnology, in particular to a single-domain heavy chain that can specifically bind to vomitoxin antigen-antibody complex (Immunocomplex) Antibody (Variable domain of heavy chain of heavy chain antibody, VHH) preparation and application.

Background technique

Immunoassay methods can be divided into competitive and non-competitive forms. Non-competitive immunoassay is widely used in the analysis of macromolecular substances containing multiple antigenic epitopes, such as microorganisms, viruses, and proteins, due to its high sensitivity and simple steps. For small molecular substances, because the molecular weight of small molecular substances is too small, it is not easy to be combined by two antibodies at the same time, so it is very difficult to develop a non-competitive mode based on double antibody sandwich for immunoassay of small molecular substances. However, in recent years, some researchers have developed a series of non-competitive immunoassay modes for small molecules based on new immune recognition components and new modes of immunoassay combinations, such as: non-competitive immunoassays based on anti-unique antibodies, Small molecular peptide heterobisite complex transfer immunoassay, non-competitive immune complex detection technology, non-competitive analysis based on chemically modified hapten, special separation equipment, special antibody immunoassay, etc., are small molecular substances The development of noncompetitive immunoassay methods provides useful exploration.

Deoxynivalneol (Deoxynivalneol, DON) is a small molecule mycotoxin of the trichothecene family, also known as vomitoxin, widely present in barley, wheat, corn, oats and other crops and their products. DON has cytotoxicity, embryotoxicity, and immunotoxicity. Mild poisoning can cause symptoms such as anorexia, vomiting, diarrhea, fever, and elevated blood pressure. In severe cases, it can threaten the lives of humans and animals. Due to the high contamination rate and high toxicity of DON, rapid detection of DON in food has important practical significance. Among the many DON detection methods, immunological detection methods have been widely used in the large-scale screening of DON in food due to their simple operation, high specificity and sensitivity. DON is a small molecular substance and cannot be combined by two conventional antibodies at the same time. As a result, most of the current immunological analysis methods for DON are based on competitive immunoassays. However, compared with the competitive immunoassay, the non-competitive immunoassay method has the advantages of less operation steps, high sensitivity, and wide detection range. Therefore, it is of great practical significance to establish a non-competitive immunoassay method for DON.

Heavy-chain antibody (Heavy-chain antibody) is a kind of antibody that naturally lacks light chain and consists only of heavy chain. It exists in animals such as camels, alpacas, sharks and cartilaginous fishes. Single-domain heavy-chain antibody, ie, nanobody (Variable domain of heavy chain of heavy-chain antibody, VHH) refers to a genetically engineered antibody consisting only of the variable region of a heavy-chain antibody. Compared with ordinary antibodies, nanobodies have the advantages of small molecular weight, good water solubility, and high stability, and have been widely used in food science research, medical diagnosis, drug development and other fields. Antigen-antibody immune complex (Immunocomplex) refers to a complex formed after the combination of antigen and antibody.

The present invention adopts phage display technology, takes the DON antigen-antibody immune complex as the target molecule, and pans the single-domain heavy-chain antibody that can specifically bind to the DON antigen-antibody immune complex from the camel-derived natural single-domain heavy-chain antibody library, On this basis, it is applied to the non-competitive immunoassay system of DON. The single-domain heavy-chain antibody that can specifically bind to the target molecule is panned by phage display technology. This method avoids the animal immunization process required for traditional antibody preparation. Noncompetitive immunoassay of DON.

Contents of the invention

The purpose of the present invention is to provide a single-domain heavy chain antibody (including proteins or polypeptides containing all or part of the functional regions of the single-domain heavy chain antibody) and its amino acid sequence that can specifically bind to the DON antigen-antibody immune complex. Used for the detection and analysis of mycotoxin DON.

The single-domain heavy chain antibody that can specifically bind to the DON antigen-antibody immune complex provided by the present invention has the amino acid sequence shown in SEQ ID NO.:1.

The IMGT numbering of its amino acid sequence and the division of structural domains are shown in Figure 1.

The single domain heavy chain antibody mentioned in the present invention includes four framework regions (Framework region, FR) and three complementarity-determining regions (Complementarity-determining region, CDR). Wherein, the framework regions (FR1-FR4) are respectively selected from SEQ ID NO.:2, SEQ ID NO.:4, SEQ ID NO.:6 and SEQ ID NO.:8, and the complementarity determining regions (CDR1-CDR3) are respectively selected from SEQ ID NO.:3, SEQ ID NO.:5 and SEQ ID NO.:7. The structure of the framework region is relatively conservative and mainly plays a role in maintaining the protein structure; the structure of the complementarity determining region is relatively diverse and is mainly responsible for the recognition of the antigen-antibody immune complex.

The present invention provides a protein or polypeptide comprising one or more amino acid sequences of SEQ ID NO.:2, SEQ ID NO.:4, SEQ ID NO.:6 and SEQ ID NO.:8, and at least It has 90% homology with one of the amino acid sequences.

The present invention provides a protein or polypeptide, comprising one or two or more of the amino acid sequences of SEQ ID NO.:3, SEQ ID NO.:5 and SEQ ID NO.:7, and at least one of the amino acid sequences 80% homology.

The present invention provides a nucleic acid molecule, which is characterized in that it encodes SEQ ID NO.: 1, and the specific sequence of the nucleic acid molecule can be obtained at any time through genetic codes.

The single domain heavy chain antibody provided by the present invention can be produced in large quantities through phage amplification. Phage amplification refers to multiplying the phage displaying the single domain heavy chain antibody through biological amplification to produce phage particles displaying the single domain heavy chain antibody.

The single-domain heavy-chain antibody provided by the present invention can be directly used for analysis and detection by phage particles displaying the single-domain heavy-chain antibody obtained through phage amplification.

The nucleotide sequence or partial sequence provided by the present invention can be expressed through a suitable expression system to obtain the corresponding protein or polypeptide. These expression systems include bacteria, yeast, filamentous fungi, animal cells, insect cells, plant cells, or cell-free expression systems.

The single domain heavy chain antibody provided by the invention can be applied to immunological detection and analysis. The types of immunological detection include enzyme-linked immunosorbent assay, colloidal gold immunochromatography, immunospot hybridization and other immunological analysis detection types based on antigen-antibody specific reaction.

The amino acid sequence provided by the present invention can be used as a precursor to be modified by random or site-directed mutation techniques to obtain mutants with better properties (water solubility, stability, affinity and specificity, etc.) for the development of further DON Protein or peptide for immunoassay.

Some terms described in the present invention have the following meanings:

Homology: Describes the degree of similarity between two or more amino acid sequences. The percentage of homology between the first amino acid sequence and the second amino acid sequence can be determined by [corresponding positions in the first amino acid sequence and in the second amino acid sequence The number of amino acid residues at the same amino acid residue] divided by [the total number of amino acids in the first amino acid sequence] and then multiplied by [100%] to calculate, wherein the deletion, insertion, Substitutions or additions (compared to the first amino acid) are considered to be differential. In addition, the percent homology can also be obtained using known computer algorithms for sequence alignment (eg, NCBI Blast).

Domain: The basic structural unit of the tertiary structure of a protein, usually with a certain function.

IMGT numbering: A standardized antibody amino acid sequence numbering method in the IMGT database (The International ImMunoGeneTics Database). For the specific numbering method, please refer to the literature (Ehrenmann, F., Q.Kaas, et al. (2010). "IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins orantibodies, T cell receptors, MHC, IgSF and MhcSF."Nucleic Acids Res 38(Database issue):D301-307.Lefranc,M.P.,C.Pommie,et al.(2003)."IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains ."Dev Comp Immunol 27(1):55-77.) description.

Codon (codon): also known as triplet code (triplet code), refers to a nucleotide triplet corresponding to a certain amino acid. The insertion of this amino acid into the growing polypeptide chain is determined during translation.

Description of drawings

Figure 1 is a schematic diagram of the amino acid numbering and structural domain of a single domain heavy chain antibody.

Fig. 2 is a non-competitive ELISA analysis DON curve based on phage display single domain heavy chain antibody. The detection range is 0-500ng/mL.

detailed description

The following will further illustrate the present invention through the panning, analysis and application of single domain heavy chain antibodies. These specific implementations should not be interpreted as limiting the scope of application of the present invention in any way.

Example 1 Affinity panning and identification of single domain heavy chain antibody specifically binding to DON antigen-antibody immune complex

The single-domain heavy chain antibody against DON antigen-antibody immune complex was screened from camel-derived natural heavy chain antibody library by solid-phase affinity panning. Purify the anti-DON mouse monoclonal antibody ascites with an affinity column to obtain the anti-DON monoclonal antibody; dilute the anti-DON monoclonal antibody to a final concentration of 50 μg/mL with PBS (pH 7.4), coat the wells of the microplate, and store at 4 °C Pack overnight. The next day, after washing 15 times with PBST (10mMPBS, 0.1% Tween-20 (v/v)), add 1% gelatin to block at 37°C for 1 hour; absorb the blocking solution, wash 5 times with PBST, and add 100 μL of DON standard to the well (20ng/mL), incubated at 37°C for 1h to form DON antigen-antibody immune complexes; then washed 5 times with PBST, and added 100μL camel-derived natural single domain heavy chain antibody library (titer about 2.0×10 ¹¹ cfu), incubated at 37°C for 1 h; washed 5 times with PBST, 7 times with sterile ddH ₂ O, added 100 μL of Glycine-HCl (0.2M, pH 2.2) to elute for 8 min, and immediately washed with 15 μL Neutralize with Tris-HCl (1M, pH 9.1); pipette the unbound phage in the well, take 10 μL to measure the titer, and use the rest to infect 25 mL of E.coli TG1 strain grown to the logarithmic phase for amplification. The conditions remained unchanged during the second, third and fourth rounds of panning, and the steps were the same as above.

After four rounds of panning, the randomly selected monoclonals were rescued with the helper phage KM13, and phage particles displaying antibody variable regions were obtained, and then the binding activity of the phage particles was determined by non-competitive phage-ELISA method. The background of the experiment was set For comparison, see Table 1 for specific sample addition steps.

Table 1 Non-competitive phage-ELISA loading table

Send the ELISA positive clones to the sequencing company for sequence determination to obtain the DNA sequence of the insert fragment, in which the sequence of the single domain heavy chain antibody is as follows:

CCATGG CCCAGTTGCAGCTCGTGGAGTCGGGTGGAGGATTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTGGCGTCGTTATGGGCTGGTTCCGCCAGGCTCTAGGGAAGGAGCGTAACTTTGTAGCGTCTATTAGCCGGAGTAGTGCATACACAAACTATGCAGACTCCGTGAAGGACCGATTCACCATCTCGAGAGACAACGCCAAGAATACGGTGTATCTGCAAATGAACAACCTGAAACCTGAGGACACGGCCCTTTATTACTGTGCAGCCGCCAACTACAGTACGACCAGAGCATCCGCGTATCGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGAACCCAAGACACCAAAACCACAA GCGGCCGC

(The underline indicates the restriction endonuclease recognition site)

According to the codon, the corresponding amino acid series is as SEQ ID NO.:1.

Example 2 Amplification of Phagemids of Single Domain Heavy Chain Antibody Specifically Binding to DON Antigen-Antibody Complex

Add the phage displaying the positive single domain heavy chain antibody to 20mL culture inoculated with E.coli TG1, shake and culture at 220rpm at 30°C for 6h; transfer the culture to another centrifuge tube, centrifuge at 4°C and 8000rpm for 15min, Transfer the supernatant to a fresh centrifuge tube, add 1/6 volume of PEG/NaCl, let stand at 4°C for 4h, centrifuge at 4°C, 8000rpm for 10min, discard the supernatant; resuspend the phage in 1mL PBS, add 1/6 After standing still at 4°C for 1 hour, centrifuge at 10,000 rpm for 10 minutes at 4°C, discard the supernatant, and add 500 μL PBS to resuspend, which is the phage amplification solution.

Example 3 Expression of a single domain heavy chain antibody specifically binding to the DON antigen antibody immune complex in Escherichia coli.

Phagemid pHEN1 was incompletely digested with restriction endonuclease NotI/NcoI, and the target fragment was recovered from agarose gel.

The obtained single-domain heavy chain antibody double-enzyme-digested gene fragment was cloned into the expression vector pET-25b, and after sequencing verification, it was named pET25b-DON.

The recombinant plasmid pET25b-DON was transformed into Escherichia coli Rosetta and induced to express. Pick a single colony and inoculate it into 5mL liquid LB/Amp medium, and culture it on a shaker at 37°C and 200rpm for 10h; inoculate the above culture solution with 1% inoculum size into 50mL liquid LB medium, shake it at 37°C and 200rpm After culturing to an OD of 0.5, a final concentration of 0.05 mM IPTG was added to induce culture at 30° C. and 180 rpm on a shaking table for 8 h.

After the induced culture was centrifuged at 8000rpm for 10min, resuspend the bacteria in 15mL of PBS and ultrasonically break, the ultrasonic condition was 220W, broken for 2s, intermittent for 3s, a total of 60 cycles; the broken bacteria were centrifuged at 4°C, 8000rpm for 15min, The supernatant was collected and subjected to affinity column purification to obtain soluble single domain heavy chain antibody.

By optimizing the induced expression conditions (such as host bacteria, expression vectors, IPTG concentration, and induction temperature, etc.), the expression of single domain heavy chain antibodies can be further increased, and it is possible to prepare a large number of single domain heavy chain antibodies that specifically bind to DON antigen-antibody complexes. Provided a way.

Example 4 Establishment of DON curve based on non-competitive ELISA analysis of phage-displayed single domain heavy chain antibody

Dilute the anti-DON monoclonal antibody to 10 μg/mL with PBS (pH 7.4) and add to the wells of the microplate, 100 μL/well, and coat overnight at 4°C; discard the coating solution, wash 3 times with 0.05% PBST, Add 300 μL of 3% skim milk, block at 37°C for 2 hours; discard the blocking solution, wash the plate 3 times with 0.05% PBST, add a series of DON standards with different concentrations in each well, 100 μL/well, incubate at 37°C for 1 hour to form DON antigen-antibody complex; discard the liquid in the well, wash the plate 3 times with 0.05% PBST, and put 100 μL of phage displaying single domain heavy chain antibody (1.0×10 ⁹ cfu)/soluble expression of single domain heavy chain antibody into each well (10 μg/mL), incubate at 37°C for 1 hour; add 1:5000 dilution of HRP-labeled anti-M13 phage secondary antibody/anti-VHH histidine-tagged secondary antibody, and incubate at 37°C for 1 hour. The color was then developed with TMB substrate and the OD ₄₅₀ was read. With the logarithm of DON concentration as the abscissa and OD ₄₅₀ as the ordinate, a non-competitive ELISA analysis DON curve based on phage display single domain heavy chain antibody was established (Fig. 2). The results showed that the single domain heavy chain antibody obtained by panning had binding activity and responsiveness to the DON antigen-antibody complex.

Claims (3)

1. can the single domain heavy chain antibody of specific binding vomitoxin Ag-Ab immune complex, there is the ammonia shown in SEQ ID NO.:1 Base acid sequence.

2. a nucleic acid molecules, it is characterized in that encoding described in claim 1 aminoacid sequence.

The application of single domain heavy chain antibody the most according to claim 1, it is characterised in that by the side that Phage amplification or genetic engineering are recombinant expressed Formula is prepared in a large number；Described Phage amplification refers to the phage that displaying has this single domain heavy chain antibody, by the way of biology expands, Amount reproduction produces shows the bacteriophage particles having this single domain heavy chain antibody；The recombinant expressed mode of described genetic engineering refers to should by coding The gene of single domain heavy chain antibody, by being cloned into expression vector, carries out a large amount of preparations of this single domain heavy chain antibody with the form of protein expression.