A soluble single-chain antibody for broad-spectrum recognition of human norovirus and its preparation method

Abstract

The present invention discloses a soluble single-chain antibody that broadly recognizes human norovirus and a preparation method thereof, relating to the field of biotechnology. The amino acid sequence of the soluble single-chain antibody is shown in SEQ ID No. 3; the preparation method comprises: obtaining the nucleotide sequence of the soluble single-chain antibody J5D that broadly recognizes hNoV; constructing a recombinant prokaryotic expression plasmid pMAL-c5X-J5D; and prokaryotic expression and purification of J5D. The amino acid sequence of the recombinant prokaryotic expression plasmid pMAL-c5X-J5D is shown in SEQ ID No. 4. The soluble single-chain antibody J5D is used in a reagent that broadly recognizes human norovirus. The present invention provides a simple and easy method for preparing a scFv that broadly recognizes different genotypes of hNoV. An scFv that broadly recognizes different genotypes of hNoV was successfully prepared, with strong specificity, high stability, and simple operation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a soluble single-chain antibody capable of broadly recognizing human norovirus and a preparation method thereof. Background Art

[0002] Currently, the main methods for detecting human norovirus (hNoV) include: molecular detection methods based on viral nucleic acid fragments and immunological methods for detecting antigenic determinants.

[0003] Molecular detection methods primarily include RT-PCR and RT-qPCR. RT-qPCR, due to its high sensitivity and specificity, has become the gold standard for hNoV detection. However, hNoV genotypes are diverse and prone to mutation, making universal detection a non-existent system. Furthermore, this method typically requires sophisticated equipment and specialized operators, with a detection cycle of approximately two hours, severely limiting its potential use cases.

[0004] Immunological methods diagnose hNoV infection (contamination) by detecting viral antigens. The major capsid protein is the primary immunogenic protein of the virus and is divided into a highly conserved capsid region and a protruding region that is prone to mutation. Targeting the protruding region can only detect a few hNoV genotypes, while targeting the conserved region allows for broad-spectrum detection. However, the monoclonal antibody preparation process used in immunological testing is complex, requires demanding technicians, is costly, and the hybridoma cells are susceptible to degeneration.

[0005] Antibodies are a type of immunoglobulin produced by effector B cells that can recognize and specifically bind to antigens. Classic antibodies have a "Y"-shaped structure consisting of two heavy chains and a light chain, both of which include variable and constant regions. Single-chain antibodies (scFv) are genetically engineered antibodies, consisting of a polypeptide chain that connects the heavy chain variable region (VH) and the light chain variable region (VL) via a linker peptide. The linker sequence should have a certain degree of hydrophilicity and should not be too short to reduce the aggregation of polypeptide chains and ensure that the linker does not affect the spatial structure formed by VH and VL during protein folding. Among them, the linker peptide (Gly4Ser)3 has good flexibility and low immunogenicity, and has become the most widely used linker.

[0006] Escherichia coli has become the most common host for scFv production due to its low cultivation cost, fast growth rate, and large number of candidate cloning vectors and host strains. When expressed in large quantities in the reducing environment of the E. coli cytoplasm, scFvs tend to form insoluble aggregates (inclusion bodies) and lack biological activity. To ensure the recognition activity of scFvs, signal peptides can be used to direct scFvs to the periplasmic space of E. coli to improve soluble expression, but the expression level is too low. In addition, adding soluble tag proteins is one of the common methods for expressing soluble single-chain antibodies. Commonly used soluble tag proteins include maltose binding protein (MBP), glutathione S-transferase (GST), and small ubiquitin-related modifier (SUMO).

[0007] Therefore, those skilled in the art are committed to developing a low-cost, soluble scFv that specifically recognizes and binds to antigens and can broadly recognize hNoVs of different genotypes. Summary of the Invention

[0008] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to prepare a low-cost, soluble scFv that specifically recognizes and binds to antigens and can broadly recognize different genotypes of hNoV.

[0009] To achieve the above objectives, the present invention provides a soluble single-chain antibody J5D, the amino acid sequence of which is shown in SEQ ID No. 3.

[0010] The present invention also provides a method for preparing a soluble single-chain antibody J5D, comprising the following steps:

[0011] Step 1: Obtain the nucleotide sequence of a soluble single-chain antibody J5D that broadly recognizes different genotypes of hNoV;

[0012] Step 2: Using the J5D gene coding sequence obtained in step 1 as a template, construct a recombinant prokaryotic expression plasmid pMAL-c5X-J5D;

[0013] Step 3: Cultivate the recombinant bacteria containing the recombinant prokaryotic expression plasmid pMAL-c5X-J5D obtained in step 2, and perform prokaryotic expression and purification of J5D.

[0014] Furthermore, step 1 further includes the following steps:

[0015] Step 1.1, design primers for the variable region of mouse monoclonal antibodies;

[0016] Step 1.2: Cultivate hybridoma cells, extract mRNA, and use the cDNA obtained by reverse transcription as a template to amplify the VH and VL gene segments of J5D using the variable region primers of the mouse monoclonal antibody obtained in step 1.1;

[0017] Step 1.3: Use the T vector containing the VH and VL gene sequences of J5D obtained in step 1.2 as templates, 5H-F and 5H-R-Linker as primers to amplify the J5D-VH-Linker nucleotide fragment, and Linker-5K-F and 5K-R as primers to amplify the Linker-J5D-VL nucleotide fragment, and splice them using SOE-PCR to obtain the J5D gene.

[0018] Furthermore, step 2 also includes extracting the recombinant prokaryotic expression plasmid pMAL-c5X-J5D, chemically transforming Origami2 competent cells, and storing positive clones for future use.

[0019] Furthermore, step 3 further includes the following steps: streaking the Origami2 Escherichia coli culture containing the recombinant prokaryotic expression plasmid pMAL-c5X-J5D on an LB solid medium containing ampicillin, and culturing; picking a single clone in an LB liquid medium containing ampicillin, and culturing with shaking; expanding the culture, and when the OD 600 When the p-value is about 0.6, take 3 mL of uninduced bacterial solution, discard the first supernatant after centrifugation, and store the first precipitate at -20°C; add IPTG to the remaining bacterial solution, culture with shaking, take the bacterial solution, discard the second supernatant after centrifugation, and store the second precipitate at -20°C; centrifuge the remaining induced bacterial solution, discard the supernatant, collect the bacteria, add Lysis buffer to resuspend, use an ultrasonic cell crusher to break the bacteria to release the protein, and collect the supernatant that may contain soluble protein and the precipitate that may contain insoluble protein respectively; the first precipitate is the uninduced bacteria, the second precipitate is the induced bacteria, the supernatant that may contain soluble protein is the supernatant after the induced bacteria are broken, and the precipitate that may contain insoluble protein is the precipitate after the induced bacteria are broken. Use SDS-PAGE to identify whether the J5D protein is successfully induced to express and whether it is soluble.

[0020] The present invention also provides a recombinant prokaryotic expression plasmid pMAL-c5X-J5D, the amino acid sequence of which is shown in SEQ ID No. 4.

[0021] Furthermore, the recombinant prokaryotic expression plasmid pMAL-c5X-J5D carries an MBP tag.

[0022] Furthermore, the MBP tag has lytic activity and can help improve the biological activity of scFv.

[0023] The present invention also provides a method for constructing a recombinant prokaryotic expression plasmid pMAL-c5X-J5D, comprising the following steps:

[0024] Step 1) Using the J5D gene coding sequence as a template, upstream primers and downstream primers are used to amplify the J5D coding gene fragment containing Nde I and Hind III restriction sites by PCR, and the fragment is recovered using an agarose gel purification kit to obtain a J5D coding gene recovery product;

[0025] Step 2) extracting the prokaryotic expression plasmid pMAL-c5X;

[0026] Step 3) digesting the recovered J5D encoding gene product obtained in step 1) and the plasmid pMAL-c5X obtained in step 2) with an endonuclease, ligating the digested J5D gene fragment and the linearized pMAL-c5X plasmid fragment using a ligase, transforming the resulting fragment into DH5α competent cells, coating a plate, and incubating the plate in an inverted manner at 37° C. to obtain a cultured plate;

[0027] Step 4) picking a single colony from the cultured plate obtained in step 3), identifying the positive clone by PCR, extracting the plasmid of the positive clone, and verifying it by DNA sequencing to obtain the recombinant prokaryotic expression plasmid pMAL-c5X-J5D;

[0028] In step 3), the endonucleases are Nde I and Hind III endonucleases, and the ligase is T4 ligase.

[0029] Furthermore, in step 1), the upstream primer contains an Nde I restriction endonuclease cleavage site, and the downstream primer contains a HindIII restriction endonuclease cleavage site and a His tag coding sequence.

[0030] Furthermore, in step 1), the upstream primer is 5H-F NdeI, the nucleotide sequence of which is shown in SEQ ID No. 5, and the downstream primer is 5K-R HindIII, the nucleotide sequence of which is shown in SEQ ID No. 6.

[0031] The present invention also provides a use of a soluble single-chain antibody J5D in a reagent for broadly identifying hNoV of different genotypes.

[0032] Furthermore, broad spectrum recognition of different genotypes of hNoV was achieved.

[0033] In preferred embodiment 1 of the present invention, a method for obtaining the nucleotide sequence of a soluble single-chain antibody that broadly recognizes hNoV is described in detail;

[0034] In another preferred embodiment 2 of the present invention, the construction process of the recombinant prokaryotic expression plasmid pMAL-c5X-J5D (scFv) is described in detail;

[0035] In another preferred embodiment 3 of the present invention, the prokaryotic expression and purification process of J5D (scFv) is described in detail;

[0036] In another preferred embodiment 4 of the present invention, the affinity determination process of J5D (scFv) is described in detail.

[0037] The beneficial technical effects of the present invention are as follows:

[0038] The present invention provides a simple and easy method for preparing a scFv that can broadly recognize different genotypes of hNoV. The scFv was successfully prepared, showing strong specificity and high stability. The major capsid protein, the primary immunogenic protein of hNoV, is divided into a highly conserved capsid region and a highly variable protruding region. The present invention provides an scFv that specifically recognizes the conserved region of the major capsid protein of hNoV.

[0039] The scFv production method proposed in this invention requires only PCR amplification to obtain the gene sequence, making it simple to operate. It can be expressed in large quantities in inexpensive E. coli, reducing production costs. It is also stable at -20°C. The scFv produced by this invention is simple to operate, significantly shortens the production cycle, reduces production costs, and exhibits excellent stability. scFv is a new type of genetically engineered antibody that, compared to monoclonal antibodies, has a smaller molecular weight, making it easier to produce and store on a large scale.

[0040] The MBP-tagged soluble scFv expressed in this invention specifically recognizes the conserved region of the major capsid protein of hNoV across different genotypes. The MBP tag was selected and expressed to express a soluble scFv while retaining its ability to specifically recognize and bind to antigens. The MBP tag has lytic activity, which can help enhance the biological activity of the scFv.

[0041] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a diagram showing the electrophoresis results of the nucleotide fragments of VH and VL according to a preferred embodiment 1 of the present invention;

[0043] Figure 2 This is a diagram showing the electrophoresis results of the J5D (scFv) gene of a preferred embodiment 1 of the present invention;

[0044] Figure 3 Schematic diagram of the plasmid map of a preferred embodiment 2 of the present invention;

[0045] Figure 4This is a graph showing the electrophoresis results of uninduced bacteria, induced bacteria, supernatant after induced bacteria were disrupted, and protein fragments precipitated after induced bacteria were disrupted, according to a preferred embodiment 3 of the present invention;

[0046] Figure 5 This is a graph showing the electrophoresis results of protein sample fragments collected by SDS-PAGE electrophoresis identification in a preferred embodiment 3 of the present invention;

[0047] Figure 6 This is a statistical result diagram of a preferred embodiment 4 of the present invention. DETAILED DESCRIPTION

[0048] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0049] Example 1 Obtaining the nucleotide sequence of a soluble single-chain antibody that broadly recognizes different genotypes of hNoV, specifically:

[0050] (1) Design of primers for the variable regions of mouse monoclonal antibodies: Specific primers were designed based on the VH and VL sequences of mouse monoclonal antibody encoding genes published in GenBank (see Table 1). Among them, 1mFH_I, 1mFH_II, 1mFH_III, 1mFH_IV, 1mFH_V, 1mFH_VI, 1mFH_VII, 1mFH_VIII, 1mFH_IX, 1mFH_X, 1mFH_XI and 1mRG*(Gamma) are primers for the first round of amplification containing VH genes; 1mFK_I, 1mFK_II, 1mFK_III, 1mFK_IV, 1mFK_V, 1mFK_VI, 1mFK_VII, 1mFK_VIII, 1mFK_IX, 1mFK_X and 1mRK* are primers for the first round of amplification containing VL (kappa) genes; 1mFL_I, 1mFL_II and 1mRL* are primers for the first round of amplification containing VL (lamda) genes. 2mFG* and 2mRG* (gamma) are primers for the second round of VH gene amplification. 2mFK* and 2mRK* are primers for the second round of VL (kappa) gene amplification. 2mFL_A*, 2mFL_B*, and 2mRL* are primers for the second round of VL (lamda) gene amplification. The linker is (GGGGS)3, and the corresponding coding nucleotide sequence is: GGTGGCGGTGGCTCGGGCGGTGGTGGATCCGGTGGCGGCGGATCT. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0051] Table 1 Primers for the variable regions of mouse monoclonal antibodies

[0052]

[0053]

[0054] (2) Amplification of J5D (scFv) VH and VL genes: Hybridoma cells were cultured, mRNA was extracted, and the cDNA obtained by reverse transcription was used as a template to amplify the VH and VL gene fragments using the specific primers listed in Table 1. The PCR reaction system was 25.0 μL: 12.5 μL of 2× PrimeSTAR GC Buffer, 2.0 μL of dNTPs, 0.5 μL each of the upstream and downstream primers (25 μM), 0.25 μL of PrimeSTAR HSDNA Polymerase (2.5 U / μL), 2.0 μL of template cDNA, and 7.25 μL of ddH2O. The amplification procedure was as follows: initial denaturation at 95°C for 3 min; 50 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. The target product was identified by 1.5% agarose gel electrophoresis (according to the gel recovery instructions provided by Thermo). The nucleotide fragments of VH and VL were both about 350 bp in size. The electrophoresis results were as follows: Figure 1 As shown, after purification and recovery, they were cloned into a T vector, and after DNA sequencing verification, they were aligned and identified as mouse VH and VL (kappa) genes on the IMGT website.

[0055] (3) Acquisition of J5D (scFv) gene

[0056] Using the T vector containing the J5D (scFv) VH and VL gene sequences as templates, 5H-F and 5H-R-Linker primers (see Table 2) were used to amplify the J5D (scFv)-VH-Linker nucleotide fragment, and Linker-5K-F and 5K-R primers were used to amplify the Linker-J5D (scFv)-VL (kappa) nucleotide fragment. These fragments were then spliced ​​using SOE-PCR. 1.5% agarose gel electrophoresis confirmed that the target product was approximately 750 bp in size. The electrophoresis results were as follows: Figure 2 As shown, DNA sequencing confirmed that it could be correctly translated, and the obtained nucleotide sequence was the J5D (scFv) gene coding sequence.

[0057] Table 2J5D (scFv) gene splicing primers

[0058]

[0059] Note: Bold represents the connecting peptide sequence

[0060] Example 2 Construction of recombinant prokaryotic expression plasmid pMAL-c5X-J5D (scFv)

[0061] Using the J5D (scFv) gene coding sequence as a template, an upstream primer containing an Nde I restriction endonuclease cleavage site and a downstream primer containing a Hind III restriction endonuclease cleavage site and a His tag coding sequence (see Table 3) were used to obtain a J5D (scFv) gene fragment containing Nde I and Hind III restriction endonuclease cleavage sites by PCR amplification, and the fragment was recovered using an agarose gel purification kit. The prokaryotic expression plasmid pMAL-c5X was extracted, and then the J5D (scFv) recovery product and plasmid pMAL-c5X were digested with NdeI and Hind III endonucleases, respectively. The digested J5D (scFv) gene fragment and the linearized pMAL-c5X plasmid fragment were ligated with T4 ligase, transformed into DH5α competent cells, spread on plates, and cultured in an inverted manner at 37°C. Single colonies were picked, positive clones were identified by bacterial liquid PCR, and plasmids were extracted. The obtained recombinant prokaryotic expression plasmid pMAL-c5X-J5D (scFv) was verified by DNA sequencing. The results showed that the sequence of J5D (scFv) was consistent with the sequence shown in SEQ ID No. 4, indicating that the recombinant prokaryotic expression plasmid pMAL-c5X-J5D (scFv) was successfully constructed. The plasmid map is shown in FIG. Figure 3 DNA sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd.

[0062] The recombinant prokaryotic expression plasmid pMAL-c5X-J5D (scFv) was extracted and chemically transformed into Origami2 (DE3) competent cells, and the positive clones were stored for future use.

[0063] Table 3J5D (scFv) construction of recombinant prokaryotic expression plasmid primers

[0064]

[0065] Note: The single-underlined base sequence is the restriction site, and the double-underlined base sequence is the coding sequence of the His tag

[0066] Example 3 Prokaryotic expression and purification of J5D (scFv)

[0067] (1) Prokaryotic expression of J5D (scFv): Take the Origami2 (DE3) Escherichia coli containing the recombinant prokaryotic expression plasmid pMAL-c5X-J5D (scFv) and streak it on LB solid medium containing ampicillin, and culture it at 37°C overnight; pick a single clone in LB liquid medium containing ampicillin, and culture it at 37°C with shaking overnight; expand the culture at a ratio of 1:100, and when the OD 600When the pH value is approximately 0.6, remove 3 mL of the uninduced bacterial culture, centrifuge, and discard the supernatant. Store the pellet at -20°C. Add IPTG (final concentration 0.6 mM) to the remaining culture, incubate at 22°C with shaking for 30 h, remove 3 mL of the culture, centrifuge, and discard the supernatant. Store the pellet at -20°C. Centrifuge the remaining induced culture at 8,000 × g for 5 min, discard the supernatant, and collect the cells. Resuspend in 5-10 volumes of Lysis Buffer (containing PMSF (final concentration 1 mM) and 0.3 mg / mL lysozyme), and disrupt the cells using an ultrasonic cell disruptor to release proteins. Ultrasonication conditions: 200 W, 4 s of disruption, 9 s of rest, for a total of 30-45 min. Centrifuge at 12,000 × g at 4°C for 20-30 min. Collect the supernatant (possibly containing soluble protein) and the pellet (possibly containing insoluble protein).

[0068] Uninduced cells, induced cells, supernatant after induced cell disruption, and precipitate after induced cell disruption were mixed with an equal volume of 2× protein loading buffer, incubated in a boiling water bath for 5 minutes, and then immediately placed in an ice bath. SDS-PAGE was used to determine whether the J5D (scFv) protein was successfully induced and expressed in a soluble form. Figure 4 As shown, the uninduced Origami2(DE3) cells harboring the recombinant prokaryotic expression plasmid pMAL-c5X-J5D(scFv) lacked the target protein band, whereas the induced Origami2(DE3) cells harboring the recombinant prokaryotic expression plasmid pMAL-c5X-J5D(scFv) contained a protein band of approximately 68 kDa, consistent with the theoretical fusion protein size, indicating successful induced expression of the J5D(scFv) protein. Both the supernatant and the precipitate after disruption of the induced cells contained the target protein, but the protein content in the supernatant was significantly higher than that in the precipitate, indicating that the J5D(scFv) recombinant protein was primarily expressed in a soluble form in Origami2(DE3) E. coli.

[0069] (2) Purification of J5D (scFv) recombinant protein: Filter the supernatant after the induced bacterial cell disruption in the previous step with a 0.45 μm filter membrane, and purify J5D (scFv) using a computer nucleic acid protein detector. The specific operation is as follows:

[0070] Load an appropriate amount of Ni NTA Beads 6FF filler into the purification column;

[0071] After filling the pump pipeline with deionized water, remove the plug on the top of the Ni column and connect it to the pump pipeline;

[0072] Remove the plug at the bottom of the Ni column and connect it to the liquid inlet tube of the computer nucleic acid protein detector, and plug it tightly to prevent leakage;

[0073] Use deionized water to flush the packing material at a rate of 1.0 mL / min for 3-5 column volumes to clean the storage solution in the Ni column;

[0074] Use Lysis Buffer to equilibrate the packing for 5 column volumes at 1.0 mL / min;

[0075] The filtered recombinant E. coli supernatant was loaded at a rate of 0.3 mL / min;

[0076] Wash 3 column volumes with Lysis Buffer at a rate of 0.3 mL / min;

[0077] Wash the residual impurities on the Ni column with 10-15 column volumes of Wash Buffer at a rate of 1.0 mL / min until the detected OD280 reaches a stable baseline;

[0078] Elute the target protein bound to the Ni column with 5-7 column volumes of Elution Buffer at a rate of 1.0 mL / min. Closely observe the OD280 value of the effluent measured by the computer nucleic acid protein detector and collect the effluent at the peak, which is the target protein.

[0079] The collected protein samples were identified by SDS-PAGE electrophoresis. Figure 5 As shown, the soluble recombinant protein J5D (scFv) was successfully purified.

[0080] Example 4 Affinity determination of J5D (scFv)

[0081] Indirect ELISA was used to detect the affinity of the soluble recombinant protein J5D (scFv) to the recombinant protein of the conserved region of the hNoV major capsid protein.

[0082] Coating: Dilute the purified soluble recombinant protein J5D (scFv) to 20 μg / mL using coating solution, add 100 μL per well to the ELISA plate, and coat overnight at 4°C. Simultaneously, coat the recombinant protein of the conserved region of the hNoV major capsid protein as a positive control, and coat with 1.0% BSA as a negative control.

[0083] The next day, the coated protein was discarded and the cells were washed three times with 150 μL PBS;

[0084] Blocking: Add 120 μL of 1.0% BSA blocking solution to each well and block at 37°C for 1 h;

[0085] The blocking solution was discarded and the cells were washed three times with 150 μL PBS;

[0086] Incubation with bound proteins: add 100 μL of hNoV major capsid protein conserved region recombinant protein to each well and incubate at 37°C for 1 h;

[0087] The bound proteins were discarded and washed three times with 150 μL TBST;

[0088] Primary antibody incubation: add 100 μL of hNoV monoclonal antibody diluted with 1.0% BSA (1:1000 dilution) to each well and incubate at 37°C for 1 h;

[0089] The primary antibody was discarded and the cells were washed three times with 150 μL TBST;

[0090] Secondary antibody incubation: add 100 μL of HRP-labeled goat anti-mouse IgG (H+L) diluted in 1.0% BSA (1:1500 dilution) to each well and incubate at 37°C for 1 h;

[0091] The secondary antibody was discarded and the cells were washed three times with 150 μL TBST;

[0092] Color development: add 100 μL TMB color development solution to each well and incubate at room temperature in the dark for 10 min;

[0093] Reaction termination: Add 50 μL of 2M H2SO4 to each well to terminate the reaction, and use a microplate reader to measure the OD value at a wavelength of 450 nm.

[0094] The statistical results are as follows Figure 6 As shown in the figure, the OD450 nm values ​​of the soluble recombinant protein J5D (scFv) and the positive control were extremely significantly higher than those of the negative control (p < 0.01), and the OD450 nm value of J5D (scFv) was extremely significantly higher than that of the positive control (p < 0.01), indicating that J5D (scFv) has a high affinity with the recombinant protein of the conserved region of the hNoV major capsid protein.

[0095] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A soluble single-chain antibody J5D, characterized in that The amino acid sequence of the soluble single-chain antibody is shown in SEQ ID No.

3.

2. The method for preparing the soluble single-chain antibody J5D according to claim 1, wherein The method comprises the following steps: Step 1: Obtain the nucleotide sequence of a soluble single-chain antibody J5D that broadly recognizes different genotypes of hNoV; Step 2: Using the J5D gene coding sequence obtained in step 1 as a template, construct a recombinant prokaryotic expression plasmid pMAL-c5X-J5D; Step 3: culture the E. coli culture medium containing the recombinant prokaryotic expression plasmid pMAL-c5X-J5D obtained in step 2, and perform prokaryotic expression and purification of J5D.

3. The preparation method according to claim 2, wherein The step 2 further comprises extracting the recombinant prokaryotic expression plasmid pMAL-c5X-J5D, chemically transforming Origami2 competent cells, and preserving positive clones for future use.

4. The preparation method according to claim 2, wherein The step 3 further includes the following steps: streaking the Origami2 Escherichia coli culture containing the recombinant prokaryotic expression plasmid pMAL-c5X-J5D on an LB solid medium containing ampicillin, and culturing; picking a single clone in an LB liquid medium containing ampicillin, and culturing with shaking; expanding the culture, and when the OD 600 When the p-value is about 0.6, 3 mL of uninduced bacterial solution was taken, the first supernatant was discarded after centrifugation, and the first precipitate was stored at -20°C; IPTG was added to the remaining bacterial solution for induction, shaking culture was performed, the bacterial solution was taken, the second supernatant was discarded after centrifugation, and the second precipitate was stored at -20°C; the remaining induced bacterial solution was centrifuged, the supernatant was discarded, the bacteria were collected, Lysis buffer was added for resuspending, and the bacteria were crushed using an ultrasonic cell crusher to release protein, and the supernatant that may contain soluble protein and the precipitate that may contain insoluble protein were collected respectively; the first precipitate was the uninduced bacteria, the second precipitate was the induced bacteria, the supernatant that may contain soluble protein was the supernatant after the induced bacteria were crushed, and the precipitate that may contain insoluble protein was the precipitate after the induced bacteria were crushed. The uninduced bacteria, the induced bacteria, the supernatant after the induced bacteria were crushed, and the precipitate after the induced bacteria were used to identify whether the J5D protein was successfully induced to express and whether it was soluble.

5. A recombinant prokaryotic expression plasmid pMAL-c5X-J5D, characterized in that: The nucleotide sequence of the recombinant prokaryotic expression plasmid is shown in SEQ ID No.

4.

6. The method for constructing the recombinant prokaryotic expression plasmid pMAL-c5X-J5D according to claim 5, wherein: The following steps are involved: Step 1) Using the J5D gene coding sequence as a template, upstream primers and downstream primers were used to obtain the Nde I and Hin The J5D encoding gene fragment at the d III restriction endonuclease cleavage site was recovered using an agarose gel purification kit to obtain a J5D encoding gene recovery product; Step 2) Extract the prokaryotic expression plasmid pMAL-c5X; Step 3) digesting the recovered J5D encoding gene product obtained in step 1) and the plasmid pMAL-c5X obtained in step 2) with an endonuclease, ligating the digested J5D gene fragment and the linearized pMAL-c5X plasmid fragment using a ligase, transforming the resulting fragment into DH5α competent cells, coating a plate, and incubating the plate upside down at 37° C. to obtain a cultured plate; Step 4) picking a single colony from the cultured plate obtained in step 3), identifying a positive clone by PCR, extracting the plasmid of the positive clone, and performing DNA sequencing verification to obtain the recombinant prokaryotic expression plasmid pMAL-c5X-J5D; The endonuclease in step 3) is Nde I and Hin d III endonuclease, and the ligase is T4 ligase.

7. The construction method according to claim 6, wherein: The upstream primer in step 1) contains Nde I restriction endonuclease enzyme cutting site, the downstream primer contains Hin dIII restriction endonuclease cutting site and His tag coding sequence.

8. The construction method according to claim 6, wherein: The upstream primer in step 1) is 5H-F Nde I, the 5H-F Nde The nucleotide sequence of I is shown in SEQ ID No.5, and the downstream primer is 5K-R Hin dIII, the 5K-R Hin The nucleotide sequence of dIII is shown in SEQ ID No.6.

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