Anti-African swine fever p72 protein single-chain antibody as well as nucleic acid molecule, recombinant expression vector, preparation method and application thereof
By designing and optimizing the expression of a single-chain antibody scFv against African swine fever p72 protein, the problems of insufficient affinity, low specificity, and poor stability in existing technologies have been solved, achieving efficient and accurate detection and prevention of African swine fever, which is applicable to various detection and drug preparation.
Patent Information
- Application Number
- CN202511826922.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-05
- Publication Date
- 2026-02-27
AI Technical Summary
Existing single-chain antibodies against African swine fever p72 protein have insufficient affinity and low specificity, are prone to cross-reaction with other porcine pathogen proteins, have poor stability, and exhibit low expression levels in E. coli expression systems, resulting in low detection sensitivity, poor accuracy, and high preparation costs.
A single-chain antibody scFv against African swine fever p72 protein was designed, consisting of a heavy chain variable region (VH), a linker peptide, and a light chain variable region (VL). High-affinity antibodies were screened using phage display technology, and efficient soluble expression was achieved in E. coli by optimizing the linker peptide sequence. A recombinant expression vector and host cells were constructed for preparation.
A single-chain antibody with high specificity, high affinity and high stability has been developed, which is suitable for rapid and accurate detection and targeted prevention of African swine fever, has the potential for large-scale production, and is applicable to a variety of detection and drug preparation.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of livestock pathogenic microorganism detection, and particularly relates to an anti-African swine fever p72 protein single-chain antibody, a nucleic acid molecule thereof, a recombinant expression vector, and a preparation method and application thereof. BACKGROUND
[0002] African swine fever is a severe infectious disease caused by African swine fever virus (ASFV) and is harmful to the pig industry. The p72 protein of the African swine fever virus is a key target for virus detection and vaccine development due to its high conservation and strong immunogenicity. At present, antibodies against the protein still mainly depend on complete monoclonal antibodies and polyclonal antibodies. The complete monoclonal antibody has a large molecular weight and poor tissue penetration, and its preparation also depends on a mammalian cell expression system, which is complex and costly, and is difficult to realize large-scale production. The polyclonal antibody has inherent defects such as low specificity and significant quality difference between different batches. These factors seriously limit the application value of existing antibodies in the precise diagnosis and efficient prevention and treatment of African swine fever.
[0003] As a small molecule antibody fragment, the single-chain antibody has the advantages of small molecular weight, strong penetration, easy expression in a prokaryotic system, and easy genetic engineering modification in theory. However, the existing single-chain antibody against the p72 protein of the African swine fever virus still has many problems. On the one hand, the affinity is generally insufficient, resulting in low sensitivity in detection application and being difficult to meet the detection needs of early diagnosis or low viral load samples. On the other hand, many single-chain antibodies still have poor specificity and are easily cross-reacted with the proteins of other common porcine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) and porcine epidemic diarrhea virus (PEDV), causing false positives in detection and seriously affecting the diagnostic accuracy. In addition, many single-chain antibodies have poor stability and are easily inactivated or have conformational changes under storage or application conditions, affecting the functional reliability. Furthermore, the existing technology has not fully solved the problem of high-efficiency soluble expression of the single-chain antibody in a prokaryotic expression system such as Escherichia coli, and the expression amount is low or the single-chain antibody exists in the form of inclusion bodies, which needs a complex refolding process, also leading to high preparation cost and complex process.
[0004] The above defects together result in that the existing single-chain antibody cannot be truly applied to the development of rapid and accurate diagnostic reagents and the development of targeted prevention and treatment products. SUMMARY
[0005] To solve the above problems in the prior art, the application aims to provide an anti-African swine fever p72 protein single-chain antibody, a detection kit and application thereof, so as to provide an antibody with high affinity, high specificity and high stability for the detection of African swine fever, and to realize rapid and accurate detection and targeted prevention and treatment of African swine fever.
[0006] To achieve the above object, the technical scheme adopted by the present application is as follows: An anti-African swine fever p72 protein single-chain antibody scFv is composed of a heavy chain variable region VH, a linker and a light chain variable region VL connected in sequence, and the amino acid sequence is shown as SEQ ID NO:1: SEQ ID NO:1 is as follows: SSSMQQSGPELVKPGASVRISCKASGYTFTSYYIHWVKQRPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARRDYGSSYGYFDVWGAGTTLTVSSGGGGSGGGGSGGGGSGGGGSDVEMTQSHKFMSTSVRDGVSITCKASQDVSTAVPWYQQKPGQSPKLLIYSASYRHTGVPDLFTGRGSGTDFTFTIISAQAEDLAEYFCQQYNSYPLSFGWGTKLEIKR The present application also provides a nucleic acid molecule encoding the anti-African swine fever p72 protein single-chain antibody.
[0007] As a limitation of the present application, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:2.
[0008] SEQ ID NO:2 is as follows: TCAAGCTCGATGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAGGATATCCTGCAAGGCTTCTGGCTACACCTTCACAAGCTACTATATACACTGGGTGAAGCAGAGGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCTTACAATGATGGAACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAGGGACTACGGTAGTAGCTACGGGTACTTCGATGTCTGGGGCGCAGGGACCACTCTCACAGTCTCCTCAGGGGGGGGGGGTAGTGGCGGCGGCGGCTCTGGAGGAGGAGGATCCGATGTTGAGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAAGAGACGGGGTCAGCATCACCTGCAAGGCCAGTCAAGATGTGAGTACTGCTGTACCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCCGCATCCTACCGGCACACTGGAGTCCCTGATCTCTTCACTGGCAGAGGATCTGGGACGGATTTCACTTTCACCATCATCAGCGCGCAGGCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATAACAGCTATCCTCTCTCGTTCGGATGGGGGACCAAGCTGGAAATAAAACGT The scFv of the application consists of a sequence of 244 amino acids, and has no stop codon inside. The heavy chain variable region and the light chain variable region can be divided into three CDR regions by analyzing the gene sequence using the IMGT website. The nucleotide sequence encoding the VH corresponds to SEQ ID NO: 2, 1-363; the nucleotide sequence encoding the Linker corresponds to SEQ ID NO: 2, 364-408; and the nucleotide sequence encoding the VL corresponds to SEQ ID NO: 2, 409-732.
[0009] The application further provides a recombinant expression vector comprising the nucleic acid molecule described above.
[0010] The present invention also provides a host cell comprising the above-described recombinant expression vector.
[0011] This invention also provides a method for preparing a single-chain antibody against African swine fever p72 protein, comprising the following steps performed sequentially: S1. Preparation of recombinant p72 protein: The gene encoding the African swine fever virus p72 protein was cloned into an expression vector, transformed into host cells, induced to express, purified, and recombinant p72 protein was obtained. S2. Construction of phage display library: Immunize animals with the recombinant p72 protein, obtain antibody-expressing cells from the immunized animals, amplify the VH and VL gene fragments of the heavy chain variable region, assemble the encoding gene of the single-chain antibody; insert the encoding gene of the single-chain antibody into the phage display vector, transform it into host cells, infect them with helper phages, and construct the phage display library; S3. Screening of specific single-chain antibodies: Using the recombinant p72 protein as the target, the phage display library was screened to obtain positive clones and determine their single-chain antibody sequences; S4. Expression and purification of single-chain antibodies: A recombinant expression vector was constructed according to the obtained single-chain antibody sequence, transformed into the expression strain for induced expression, purified, and obtained a single-chain antibody against African swine fever p72 protein.
[0012] As a further limitation of the present invention, the expression vector is pET-28a; the linker peptide is (Gly4Ser)3 linker peptide; the phage display vector is pComb3XSS; and the helper phage is M13KO7.
[0013] As a further limitation of the present invention, in step S2, the cells expressing the antibody are spleen cells; total RNA is extracted from spleen cells and reverse transcribed into cDNA, which is used as a template for overlap extension PCR.
[0014] The present invention also provides a detection reagent comprising a single-chain antibody against African swine fever p72 protein prepared by the above preparation method.
[0015] The present invention also provides the application of the above-mentioned anti-African swine fever p72 protein single-chain antibody in the preparation of products for detecting African swine fever virus or targeted anti-African swine fever virus drugs.
[0016] Preferably, the products for detecting African swine fever virus include flow test strips and enzyme-linked immunosorbent assay (ELISA) kits.
[0017] By adopting the above-described technical solution, the beneficial effects achieved by this invention compared to the prior art are as follows: (1) The anti-African swine fever p72 protein single-chain antibody provided by the application has high specificity for the African swine fever virus p72 protein, and no cross-reaction is observed in a variety of porcine pathogen protein detection systems including porcine reproductive and respiratory syndrome virus and porcine epidemic diarrhea virus N protein, and is suitable for the development of high-specificity diagnostic reagents.
[0018] (2) The single-chain antibody sequence with extremely high affinity for the p72 protein is obtained through directional screening of the phage display technology, which provides a basis for constructing a high-sensitivity detection system and helps to improve the performance index of the detection reagent.
[0019] (3) The optimized (Gly4Ser) 3 connecting peptide sequence is used in the application, so that the single-chain antibody maintains a stable spatial conformation and efficient soluble expression in the Escherichia coli expression system, and the expression amount reaches about 5 mg of soluble recombinant protein per 1L of Escherichia coli cells under laboratory conditions, which has the feasibility of large-scale production.
[0020] (4) The anti-African swine fever p72 protein single-chain antibody of the application can be combined with a variety of reporter molecules such as fluorescent proteins and enzyme labels to construct fusion proteins through genetic engineering methods, and is suitable for the preparation of a variety of detection kits such as lateral flow test strips and enzyme-linked immunoassay and target anti-African swine fever virus drugs. BRIEF DESCRIPTION OF DRAWINGS
[0021] The application will be described in further detail below in combination with the drawings and specific embodiments.
[0022] Figure 1 It is a purification result graph of the recombinant ASFV p72 protein in the embodiment 1 of the application; Figure 2 It is a serum antibody titer determination result graph of the mice immunized in the embodiment 1 of the application; Figure 3 It is a gene amplification result graph in the scFv phage display library construction process in the embodiment 1 of the application; Figure 4 It is a result graph of the growth of colonies obtained by coating plates with the bacterial liquid diluted to 10 -5 and 10 -6 in the quality identification of the scFv phage display library in the embodiment 1 of the application; Figure 5 It is a colony PCR verification result graph in the quality identification of the scFv phage display library in the embodiment 1 of the application; Figure 6 It is a detection result graph of the detection of the binding capacity of Nb17 and the recombinant p72 protein by indirect ELISA in the effect experiment example of the application; Figure 7Figure of the results of the detection of the specificity of Nb17 by indirect ELISA in the effect experiment of the present application. DETAILED DESCRIPTION
[0023] The preferred embodiments of the present application will be described in detail below with reference to the drawings. It should be understood that the preferred embodiments described herein are merely intended to illustrate and explain the present application, and are not intended to limit the present application.
[0024] The materials, reagents, etc. used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified. The experimental methods not specified in the embodiments are usually performed according to conventional conditions or according to the recommended conditions of the manufacturers.
[0025] Example 1 The present embodiment is a method for preparing a single-chain antibody against African swine fever p72 protein, which comprises the following steps performed in sequence: S1. Preparation of recombinant p72 protein The coding gene of African swine fever virus (ASFV) p72 protein (derived from GenBank accession number MN886926.1) was cloned into the prokaryotic expression vector pET-28a(+) to construct a recombinant expression vector pET-28a-p72. The recombinant vector was transformed into E. coli BL21 competent cells, and 0.8 mM IPTG was used to induce expression at 16°C for 18 hours to promote the production of soluble protein. After collecting the bacterial cells, the expression product was purified by a Ni²⁺-NTA affinity chromatography column. The purity of the recombinant p72 protein was verified by SDS-PAGE electrophoresis, as shown in Figure 1 The size of the target protein is about 72 kDa. As Figure 1 shown in the left graph, the target protein purified by the nickel column started to fall off in a 40 mM imidazole solution but mixed with some impure proteins, and a small amount of the target protein was relatively pure in a 60 mM imidazole solution. After the target protein was collected and concentrated by ultrafiltration, it was verified by SDS-PAGE (as Figure 1 shown in the middle graph) and western blot (as Figure 1 shown in the right graph), and the purity of the obtained target protein reached more than 90% except for a small amount of protein degradation. The concentration was determined, and the obtained product was the high-purity recombinant ASFV p72 protein used for subsequent immunization and screening.
[0026] S2. Construction of phage display library Balb / c mice aged 6-8 weeks were immunized with the high-purity recombinant p72 protein prepared in step S1 as an antigen. The first immunization was performed by intraperitoneal injection of 150 μg of the antigen emulsified with an equal volume of Freund's complete adjuvant. Thereafter, booster immunization was performed once every two weeks for a total of three times. The last immunization was performed without the addition of an adjuvant. Three days after the final immunization, the serum titer of the mice was measured, and, as shown in Table 1, the immunization effect was confirmed to be good. Figure 2 After the immunization effect was confirmed to be good, the mice were sacrificed and their spleens were removed to prepare a spleen cell suspension by grinding and filtration.
[0027] Total RNA was extracted from the spleen cells and reverse-transcribed to synthesize cDNA. The cDNA was used as a template to perform PCR amplification using specific primers for the heavy chain variable region (VH) and the light chain variable region (VL) as shown in Tables 1 and 2, respectively, to obtain the VH and VL gene fragments. Figure 3 As shown in Table 1, the heavy chain variable region fragments were amplified by respective VH FOR primers and different VH REV primers. The κ light chain variable region fragments were amplified by respective VK FOR primers and VK REV primers. The λ light chain variable region was amplified by respective Vλ FOR primers and Vλ REV primers. After all of the cDNAs were equally divided, they were used as templates to perform a total of 116 amplifications. The VH and VL fragments were about 370 bp in length, and the target bands were located between 200 bp and 500 bp markers. The obtained light chain variable region and heavy chain variable region were used as primers to perform overlap PCR to amplify the complete scFv fragment, and the scFv fragment was about 750 bp in length. The target bands were located at about 750 bp markers to construct the complete single-chain antibody (scFv) coding gene.
[0028] Table 1 VH-specific primer sequences Table 2 VL-specific primer sequences The obtained scFv gene was cloned into the phage display vector pComb3XSS, and then the recombinant vector was introduced into E. coli TG1 competent cells by electroporation. After infection with the helper phage M13KO7, the scFv phage display library was successfully constructed.
[0029] The library capacity of the constructed library was measured by plate counting. After activation, a small amount of the bacterial solution obtained by electroporation was gradient-diluted, and the bacterial solution was plated to measure the library capacity according to the number of colonies. The library capacity of the immune library was about 2.6 x 1010 7 cfu / mL, as shown in Table 2. Figure 4 The bacterial solution was diluted 10-5 and 10 -6 The colonies grown on the post-coated plates were counted and the number of colonies was calculated as follows: Figure 5 The randomly picked clones were verified by colony PCR, and the insertion positive rate was about 80%, indicating that the library quality was good and could be used for subsequent screening.
[0030] S3. Screening of specific single-chain antibodies The recombinant p72 protein prepared in step S1 was used as the immobilized target to perform solid-phase biopanning on the phage display library constructed in step S2 to screen specific single-chain antibodies with high affinity. The specific process was as follows: The enzyme-labeled plate was coated with 5 μg / mL of recombinant p72 protein at 4°C overnight; after blocking, the appropriate dilution of phage library was added, and incubated at 37°C for 2 hours; then PBST and PBS were used for strict washing to remove unbound or non-specifically bound phages; TG1 host bacteria solution was added to recover the bound phages for the next round of amplification.
[0031] Three rounds of screening were performed, and the strictness of the screening was increased in subsequent rounds by reducing the antigen coating concentration to 2 μg / mL and increasing the number of PBST washes to 15 to enrich high-affinity clones.
[0032] After each round of screening, the OD450nm absorbance value was detected by phage ELISA to evaluate the enrichment effect. As shown in Table 3, the signal value increased significantly with the rounds, indicating that specific phages were effectively enriched.
[0033] Table 3. Enrichment effect table of three rounds of screening After completing three rounds of screening, 20 single clones were selected from the culture plate of the third round of biopanning, and soluble recombinant nanobody crude extract was prepared by IPTG induction. The reactivity of the recombinant antibody crude extract with the p72 recombinant protein was preliminarily identified by recombinant antibody crude extract ELISA. The specific reaction steps were as follows: (1) Each clone was inoculated into an EP tube containing 1 mL / well of LB medium, and cultured at 37°C and 180 r / min until the logarithmic phase (OD600 reached 0.6-0.8). IPTG with a final concentration of 1 mM was added, and the induction was performed at 37°C and 180 r / min overnight. The induced product was centrifuged at 4°C and 3500 g for 10 min, and the bacteria were collected and the supernatant was discarded. The bacteria were repeatedly frozen and thawed three times at -80°C. 200 μL of PBS buffer was added to each tube to resuspend the bacteria, and centrifuged at 4°C and 7000 rpm / min for 15 min. The supernatant (containing soluble recombinant nanobody crude extract) was collected and used.
[0034] (2) Coating liquid dilutes p72 recombinant protein to 3 μg / mL, 100 μL / well is added to a 96-well plate, PBS is used as a blank control, and coating is performed at 4°C overnight.
[0035] (3) 150 μL / well of blocking solution is added, and blocking is performed at 37°C for 1 h.
[0036] (4) The blocking solution is discarded, and the soluble recombinant nanobody crude extract (dilution ratio of 1:1) is added to the plate at 100 μL / well, and incubation is performed at 37°C for 1 h.
[0037] (5) 100 μL / well of mouse anti-HA tag monoclonal antibody (dilution ratio of 1:2000) is added, and incubation is performed at 37°C for 1 h, and plate washing is performed three times with PBST.
[0038] (6) 100 μL / well of HRP-labeled goat anti-mouse monoclonal antibody (dilution ratio of 1:2000) is added, and incubation is performed at 37°C for 1 h, and plate washing is performed three times with PBST.
[0039] (7) 100 μL / well of color developing solution is added, and reaction is performed at room temperature for 15 min in the dark. 50 μL / well of sulfuric acid solution is added to terminate the reaction, and the OD450 value of each well is determined using a multifunctional enzyme marker.
[0040] Each group of reactions is repeated three times, and a negative control is set. Finally, the OD450 value greater than three times the value of the negative control is determined as a positive clone, and the strain with a higher value is selected for sequencing. Finally, a single-chain antibody gene sequence with high affinity is obtained, which is named Nb17 according to its number. The amino acid sequence encoded by Nb17 is SEQ ID NO: 1, and the nucleotide sequence thereof is SEQ ID NO: 2.
[0041] S4. Expression and purification of single-chain antibody According to the Nb17 gene sequence obtained by sequencing in step S3, a primer is designed, and a recombinant expression vector pET-28a-Nb17-HA is constructed, that is, an HA tag sequence is fused to the C-terminal end of Nb17 and a stop codon is added. The recombinant plasmid is transformed into an Escherichia coli BL21 expression strain. Referring to the induction expression condition (0.8 mM IPTG, 16°C, 18 hours) in step S1, the single-chain antibody Nb17-HA is expressed in large quantities. After collecting the bacterial cells, ultrasonic crushing, centrifugation, and taking the supernatant, nickel column affinity chromatography is used for purification. The purified protein is concentrated by ultrafiltration, the concentration is about 3.2 mg / mL as determined by a Nanodrop protein quantifier, and the protein is stored at -80°C after being aliquoted for standby, that is, the anti-African swine fever p72 protein single-chain antibody of the application.
[0042] Effect experiment example 1 The effect experiment example detects the binding ability of single-chain antibody and p72 protein by indirect ELISA, and the specific steps are as follows: The recombinant p72 protein is diluted with coating solution to 2 μg / mL, 100 μL / well is added to the 96-well plate, and is coated at 4°C overnight. After washing the plate with PBST for 3 times, 150 μL / well of blocking solution (PBST containing 5% skimmed milk) is added, and is blocked at 37°C for 1 hour.
[0043] The blocking solution is discarded, and different dilution ratios (1:1, 1:10, 1:100, 1:1000) of purified single-chain antibody Nb17 solution are added, 100 μL / well, and are incubated at 37°C for 1 hour. After washing the plate with PBST for 3 times, mouse anti-HA IgG (1:2000 dilution) is added as the secondary antibody, and is incubated at 37°C for 1 hour; then HRP-labeled goat anti-mouse IgG (1:2000 dilution) is added as the tertiary antibody, and is incubated at 37°C for 1 hour, and the plate is washed with PBST for 3 times after each incubation.
[0044] 100 μL / well of TMB color developing solution is added, and is reacted at room temperature for 15 minutes in the dark, 50 μL / well of 2 M H2SO4 is added to terminate the reaction, and the OD450 value is determined by using a multifunctional enzyme label instrument.
[0045] The results are shown in Figure 6 The Nb17 antibody can produce a significant signal with the p72 protein at each dilution gradient, and the OD450 value decreases in a gradient manner as the antibody concentration decreases, showing a good dose-dependent relationship, indicating that the single-chain antibody has a strong antigen binding ability.
[0046] Effect experiment example 2 The effect experiment example is used for specific detection of single-chain antibody. The specific steps are as follows: In order to verify the specificity of Nb17, porcine epidemic diarrhea virus (PEDV) N protein and porcine reproductive and respiratory syndrome virus (PRRSV) N protein are selected as control antigens. The recombinant p72 protein, PEDV N protein and PRRSV N protein are diluted to 2 μg / mL, 100 μL / well is added to the same 96-well plate, and is coated at 4°C overnight.
[0047] The remaining steps are the same as those in experiment example 1, and the same concentration of Nb17 antibody is added for reaction. The results are shown in Figure 7 The Nb17 antibody can produce a significant signal with the p72 protein at each dilution gradient, and the OD450 value decreases in a gradient manner as the antibody concentration decreases, showing a good dose-dependent relationship, indicating that the single-chain antibody has a strong antigen binding ability.
[0048] The results show that the Nb17 single-chain antibody can specifically recognize ASFV p72 protein, and does not cross-react with other common porcine virus proteins, and has high specificity.
[0049] Embodiment 2 The present application provides a detection reagent, the core component of which is the anti-African swine fever p72 protein single-chain antibody Nb17 prepared by the method described in the aforementioned embodiment 1. The detection reagent can be used to construct various forms of diagnostic products.
[0050] For example, taking the preparation of an enzyme-linked immunosorbent assay (ELISA) kit for detecting African swine fever virus as an example, the preparation process is as follows: the purified single-chain antibody Nb17 is used as a coating antibody or a detection antibody. Specifically, Nb17 is coated in 96-well polystyrene enzyme-coated plates at an appropriate concentration, such as 1-5 µg / mL, at 4°C overnight, and after blocking and drying, a solid-phase antibody reaction plate is prepared.
[0051] Other components of the kit also include: sample diluent, washing buffer, blocking solution, HRP-labeled Nb17, color developing substrate solution (such as TMB), and reaction termination solution.
[0052] All components are aseptically packaged and assembled according to the conventional process to obtain a complete ELISA detection reagent kit prototype. The kit uses the principle of double-antibody sandwich method and can specifically capture and detect ASFV p72 antigens in the sample.
[0053] In other embodiments, the kit contains Nb17 labeled with a reporter molecule, which can be obtained by genetically engineering Nb17 to be expressed in fusion with HRP.
[0054] Embodiment 3 The present application also provides the use of the aforementioned anti-African swine fever p72 protein single-chain antibody in the preparation of products for detecting African swine fever virus, specifically for the preparation of lateral flow immunochromatographic test strips for rapid on-site diagnosis.
[0055] The preparation method is as follows: the purified single-chain antibody Nb17 is used as the immobilized antibody of the test line (T line), and the antibody against the HA tag or other universal tag is used as the immobilized antibody of the control line (C line), which are respectively sprayed on the corresponding positions of the nitrocellulose membrane. Another anti-p72 protein monoclonal antibody (or the labeled Nb17 itself) labeled with colloidal gold particles is freeze-dried on a conjugate pad. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad are sequentially assembled on a back plate and cut into strips. When used, the sample to be tested, such as tissue homogenate or serum, is added to the sample pad, and through chromatography, the ASFV p72 antigens in the sample will bind with the gold-labeled antibody, and the complex formed will continue to chromatograph to the T line, where it will be captured by the immobilized Nb17 and accumulate to develop color; whether the sample contains antigens or not, the unbound gold-labeled antibody will be captured by the C line to develop color, thereby achieving the determination of the results. This method is simple and rapid and is suitable for on-site screening at the grassroots level.
[0056] In addition, the single-chain antibody also has application potential in the preparation of drugs targeting against African swine fever virus. For example, the coding gene of Nb17 can be fused with the coding gene of a protein having cell penetration function or toxin to construct a recombinant expression vector. The vector is introduced into a suitable expression system, such as a mammalian cell, for expression, so as to produce a fusion protein having the ability of targeting ASFV infected cells. The fusion protein can specifically bind to the virus p72 protein by means of Nb17, and direct the delivery of functional proteins, such as toxins or enzymes inhibiting virus replication, to infected cells or virus particles, so as to exert a therapeutic effect. This application provides a new idea and candidate molecules for the development of new biological agents against ASFV.
[0057] It should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art can modify the technical solutions described in the above embodiments or make equivalent replacements to some technical features, and any equivalent replacement shall be included in the protection scope of the present application.
Claims
1. A single-chain antibody against African swine fever p72 protein, comprising a heavy chain variable region VH, a linker peptide, and a light chain variable region VL connected sequentially, characterized in that, Its amino acid sequence is shown in SEQ ID NO:
1.
2. A nucleic acid molecule, characterized in that, It encodes the anti-African swine fever p72 protein single-chain antibody as described in claim 1.
3. The nucleic acid molecule according to claim 2, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:
2.
4. A recombinant expression vector, characterized in that, It comprises the nucleic acid molecule as described in claim 2 or 3.
5. A host cell, characterized in that, It comprises the recombinant expression vector as described in claim 4.
6. A method for preparing the single-chain antibody against African swine fever p72 protein as described in claim 1, characterized in that, This includes the following steps performed sequentially: S1. Preparation of recombinant p72 protein: The gene encoding the African swine fever virus p72 protein was cloned into an expression vector, transformed into host cells, induced to express, purified, and recombinant p72 protein was obtained. S2. Construction of phage display library: Immunize animals with the recombinant p72 protein, obtain antibody-expressing cells from the immunized animals, amplify the VH and VL gene fragments of the heavy chain variable region, assemble the encoding gene of the single-chain antibody; insert the encoding gene of the single-chain antibody into the phage display vector, transform it into host cells, infect them with helper phages, and construct the phage display library; S3. Screening of specific single-chain antibodies: Using the recombinant p72 protein as the target, the phage display library was screened to obtain positive clones and determine their single-chain antibody sequences; S4. Expression and purification of single-chain antibodies: A recombinant expression vector was constructed according to the obtained single-chain antibody sequence, transformed into the expression strain for induced expression, purified, and obtained a single-chain antibody against African swine fever p72 protein.
7. The method for preparing the anti-African swine fever p72 protein single-chain antibody according to claim 6, characterized in that, The expression vector is pET-28a; the linker peptide is (Gly4Ser)3 linker peptide; the phage display vector is pComb3XSS; and the helper phage is M13KO7.
8. The method for preparing the anti-African swine fever p72 protein single-chain antibody according to claim 6 or 7, characterized in that, In step S2, the cells expressing the antibody are spleen cells; total RNA is extracted from spleen cells and reverse transcribed into cDNA, which is used as a template for overlap extension PCR.
9. A detection reagent, characterized in that, It comprises the anti-African swine fever p72 protein single-chain antibody prepared by any one of the preparation methods of claims 6 to 8.
10. The use of the anti-African swine fever p72 protein single-chain antibody according to claim 1 in the preparation of products for detecting African swine fever virus or targeted anti-African swine fever virus drugs.