Anti-Muscovy duck parvovirus VP3 protein recombinant nano antibody, expression engineering strain and application thereof
By employing computational biology and molecular docking screening methods, the variable region sequence VP3-Nb66 of the heavy chain antibody was constructed. A highly efficient and low-cost recombinant nanobody against Muscovy duck parvovirus VP3 protein was prepared using the Pichia pastoris expression system. This approach overcomes the shortcomings of traditional antibodies in the preparation and expression process, enabling large-scale detection at a high efficiency and low cost.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-18
- Publication Date
- 2026-04-03
AI Technical Summary
Traditional monoclonal antibodies in existing technologies suffer from long preparation and expression cycles, high costs, limited stability, large molecular weight, and weak tissue penetration, making it difficult to meet the needs of large-scale detection. In particular, there is a lack of nanobodies with high affinity and high expression levels for the detection of Muscovy duck parvovirus VP3 protein.
Using computational biology-aided design and molecular docking screening, the variable region sequence VP3-Nb66 of a heavy chain antibody was constructed, and recombinant nanobodies were prepared using a Pichia pastoris expression system. High-affinity and low-cost nanobodies were prepared by using recombinant eukaryotic expression plasmids and engineered strains for efficient expression.
A recombinant nanobody with high binding activity against Muscovy duck parvovirus VP3 protein was obtained, with a yield of 1.01 mg/L. This nanobody formulation is suitable for large-scale production, simplifying the operation process and reducing costs.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, and in particular relates to a recombinant nanobody against Muscovy duck parvovirus VP3 protein, an engineered strain expressing the nanobody, and their applications. Background Technology
[0002] Muscovy duck parvovirus (MDPV) is the main pathogen causing Muscovy duck parvovirus disease. This disease is characterized by acute, highly contagious infection, and clinical manifestations include severe enteritis, myocarditis, and immunosuppression. It has an extremely high mortality rate in young Muscovy ducks, causing significant economic losses to the Muscovy duck farming industry. VP3 protein is one of the main structural proteins of Muscovy duck parvovirus and plays a crucial role in viral assembly, infection, and immune recognition, thus becoming an important target for diagnosis and immune intervention.
[0003] Currently, detection methods for Muscovy duck parvovirus mainly include virus isolation, PCR technology, and serological detection (such as ELISA). Among these, serological detection is widely used due to its simplicity and suitability for large-scale screening, but its core relies on high-quality specific antibodies. While traditional monoclonal antibodies have advantages such as high specificity and good uniformity, they suffer from problems such as long preparation cycles, high costs, limited stability, and difficulty in efficient expression in prokaryotic or eukaryotic systems. Furthermore, traditional antibodies have large molecular weights and weak tissue penetration, limiting their application in certain detection scenarios.
[0004] In recent years, nanobodies (Nb), as variable-region single-domain antibodies derived from camel heavy chain antibodies, have shown great promise in disease diagnosis, treatment, and research tool development due to their advantages such as small molecular weight, structural stability, good solubility, ease of genetic engineering, and large-scale expression. However, research on nanobodies targeting the Muscovy duck parvovirus VP3 protein is still in its early stages, and there is a severe lack of publicly reported anti-VP3 nanobodies with high affinity, high expression levels, and suitability for large-scale production.
[0005] Therefore, developing a specific nanobody with high affinity, easy expression, low cost, and suitable for detecting Muscovy duck parvovirus is of great significance for improving the diagnostic efficiency of this disease and promoting the development of related diagnostic reagents. Summary of the Invention
[0006] The purpose of this invention is to provide a recombinant nanobody against Muscovy duck parvovirus VP3 protein, an engineered strain expressing the nanobody, and their applications.
[0007] This invention aims to obtain a novel recombinant nanobody against Muscovy duck parvovirus VP3 protein by combining computational biology-aided design, molecular docking screening, and efficient expression system construction, in order to overcome the shortcomings of existing technologies.
[0008] The technical solution adopted by this invention to solve the technical problem is as follows:
[0009] In a first aspect, the present invention provides a heavy chain antibody variable region sequence VP3-Nb66.
[0010] Furthermore, the amino acid sequence of the variable region sequence VP3-Nb66 of the heavy chain antibody is SEQ ID NO:2.
[0011] Furthermore, the nucleotide sequence encoding the amino acid sequence of the variable region sequence VP3-Nb66 of the heavy chain antibody is SEQ ID NO:1.
[0012] Secondly, the present invention provides a method for screening a heavy chain antibody variable region sequence VP3-Nb66, comprising the following steps:
[0013] A computer-simulated molecular docking screening method was adopted. AlphaFold3 was used for high-precision 3D modeling of the antigen, RFdiffusion was used for antibody design backbone, and Proteinmpnn was used for sequence design. Using Muscovy duck parvovirus VP3 protein antigen as ligand, global docking calculations were performed. The optimal binding energy between the nanobody and the antigen was -319.33 kcal / mol, thus obtaining a heavy chain antibody variable region sequence VP3-Nb66.
[0014] Thirdly, the present invention provides a recombinant eukaryotic expression plasmid constructed using the variable region sequence VP3-Nb66 of the aforementioned heavy chain antibody.
[0015] Furthermore, the nucleotide sequence of the recombinant eukaryotic expression plasmid is SEQ ID NO:3.
[0016] Fourthly, the present invention provides a method for constructing the recombinant eukaryotic expression plasmid, comprising the following steps:
[0017] The heavy chain antibody variable region sequence VP3-Nb66 and the pPIC9K vector were digested with restriction endonucleases EcoRI and NotI, respectively, and then recovered by 1% agarose gel electrophoresis. The target fragment was ligated with T4 ligase and transformed into E. coli DH5α competent cells. The cells were plated and cultured overnight. The next day, single colonies were picked and sequenced. The correctly identified recombinant eukaryotic expression plasmid was named pPIC9K-DPV-VP3-Nb66.
[0018] Fifthly, the present invention provides a recombinant expression engineered strain constructed using the recombinant eukaryotic expression plasmid described above.
[0019] Sixthly, the present invention provides a method for constructing the recombinant expression engineered strain, comprising the following steps:
[0020] The recombinant eukaryotic expression plasmid pPIC9K-DPV-VP3-Nb66 was linearized using the restriction endonuclease Sal I, added to Pichia pastoris X-33 competent cells, mixed well, and then transferred to a pre-chilled electroporation cuvette. After being placed on ice, the cuvette was transferred to an electroporator for electroporation. After electroporation, pre-chilled sorbitol was added, and the cuvette was pipetted and transferred to a centrifuge tube. The cuvette was incubated statically at 30°C, centrifuged at room temperature, and the cells were collected and resuspended in YPG medium. The cells were then spread onto YPG solid medium containing bleomycin and incubated at 37°C for 2-3 days. Single colonies were picked for PCR identification. If the identification was correct, the recombinant expression engineered strain was obtained.
[0021] In a seventh aspect, the present invention provides a recombinant nanobody against Muscovy duck parvovirus VP3 protein prepared using the aforementioned recombinant expression engineered strain.
[0022] Eighthly, the present invention provides the use of the recombinant nanobody against Muscovy duck parvovirus VP3 protein in the preparation of nanobody formulations for detecting Muscovy duck parvovirus disease.
[0023] The beneficial effects of this invention are:
[0024] The recombinant nanobody against Muscovy duck parvovirus VP3 protein of the present invention was obtained by screening using computational biology, molecular docking simulation, and deep learning techniques. First, a heavy chain antibody variable region sequence VP3-Nb66 was screened. Then, a recombinant eukaryotic expression plasmid was constructed using this heavy chain antibody variable region sequence VP3-Nb66. Subsequently, a recombinant expression engineered strain was constructed using this recombinant expression engineered strain, and finally, a recombinant nanobody against Muscovy duck parvovirus VP3 protein was prepared using this recombinant expression engineered strain.
[0025] The recombinant nanobody against Muscovy duck parvovirus VP3 protein of the present invention was prepared by a Pichia pastoris expression system, and its yield reached 1.01 mg / L.
[0026] The ELISA results of the recombinant nanobody against Muscovy duck parvovirus VP3 protein of the present invention are as follows: 450 =1.66, exhibiting significant binding activity, and can be used to prepare novel nanobody formulations for detecting Muscovy duck parvovirus disease.
[0027] The present invention provides a method for preparing recombinant nanobodies against Muscovy duck parvovirus VP3 protein, which is simple to operate, low in cost, and can ensure the yield, activity, safety and production applicability of the recombinant nanobodies against Muscovy duck parvovirus VP3 protein, and is suitable for large-scale production. Attached Figure Description
[0028] Figure 1This is a map of the recombinant eukaryotic expression plasmid pPIC9K-DPV-VP3-Nb66 in Example 2 of this invention.
[0029] Figure 2 The expression results of the recombinant nanobody VP3-Nb66 in Example 4 of this invention are shown.
[0030] Figure 3 The results show the binding activity detection of the recombinant nanobody VP3-Nb66 in Example 5 of this invention. Detailed Implementation
[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0032] Example 1: Computer simulation of molecular docking to screen a heavy chain antibody variable region sequence (VHH sequence).
[0033] High-precision 3D modeling was performed using AlphaFold3 against the antigen, antibody backbone design was conducted using RFdiffusion, and sequence design was performed using Proteinmpnn. Global docking calculations were performed using the Muscovy duck parvovirus VP3 protein antigen as a ligand. The optimal binding energy between the nanobody and the antigen was -319.33 kcal / mol, thus obtaining a heavy chain antibody variable region sequence (VHH sequence) VP3-Nb66. The nucleotide sequence of VP3-Nb66 is SEQ ID NO:1, and the encoded amino acid sequence is SEQ ID NO:2.
[0034] Example 2 Construction and Identification of Recombinant Eukaryotic Expression Plasmids
[0035] The VHH sequence (VP3-Nb66) screened in Example 1 and the pPIC9K vector (purchased from Thermo Fisher Scientific (China) Co., Ltd.) were digested with restriction endonucleases EcoRI and Not I (both EcoRI and Not I were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.), respectively. The fragments were recovered by 1% agarose gel electrophoresis. The target fragment was ligated with T4 ligase (purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.) and transformed into E. coli DH5α competent cells (purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.). The cells were plated and cultured overnight. The next day, single colonies were picked, and the correctly identified recombinant eukaryotic expression plasmid was named pPIC9K-DPV-VP3-Nb66 (…). Figure 1Store at -20℃ for later use. Its nucleotide sequence is SEQ ID NO:3.
[0036] Example 3 Construction and Identification of Recombinant Expression Engineered Strains
[0037] The recombinant eukaryotic expression plasmid pPIC9K-DPV-VP3-Nb66 obtained in Example 2 was linearized using the restriction endonuclease Sal I, added to Pichia pastoris X-33 competent cells, mixed well, and transferred to a pre-cooled electroporation cuvette. After being placed on ice, it was transferred to an electroporator for electroporation. After electroporation, pre-cooled sorbitol was added, and the mixture was pipetted and transferred to a centrifuge tube. The cells were incubated statically at 30°C, centrifuged at room temperature, and the cells were collected and resuspended in YPG medium. The cells were then spread onto YPG solid medium containing bleomycin and incubated at 37°C for 2-3 days. Single colonies were picked for PCR identification. If the identification was correct, the recombinant expression engineered strain was obtained.
[0038] Example 4: Preparation of recombinant nanobody VP3-Nb66
[0039] The recombinant expression strain obtained in Example 3 was first inoculated into 20 mL of YPG medium for rejuvenation. The next day, 5 mL was inoculated into a 1 L shake flask (containing 250 mL of YPG medium) and cultured overnight at 28°C and 200 r / min. The bacterial cells were collected by centrifugation and resuspended in an equal volume of BMMY liquid medium (purchased from Beijing Solarbio Science & Technology Co., Ltd.). The cells were induced at 28°C and 200 r / min for 120 hours (with methanol added every 24 hours to a final concentration of 0.5%). The supernatant was collected and purified according to the affinity chromatography column (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) instructions to obtain the recombinant protein. The molecular weight and purity of the recombinant protein were analyzed by SDS-PAGE and Western blot. The SDS-PAGE results are as follows: Figure 2 As shown, the relative molecular mass of the obtained recombinant protein is 14.6 kDa, which is consistent with the expected value, indicating that the recombinant protein is the recombinant nanobody VP3-Nb66, with a concentration of 1.01 mg / L and a purity of 70%.
[0040] Example 5: Detection of the binding activity of recombinant nanobody VP3-Nb66
[0041] The binding activity of the recombinant nanobody VP3-Nb66 was verified by ELISA. Muscovy duck parvovirus was diluted with 0.05 mol / L, pH 9.6 carbonate buffer and coated at a concentration of 1 μg / well. 10% fetal bovine serum (NEWZERUM) was used as the blocking buffer. The primary antibody was the recombinant nanobody VP3-Nb66, and the secondary antibody was an anti-His-HRP tag antibody (Thermo Fisher Scientific). TMB was added for color development, and the reaction was terminated with 2M H2SO4. OD was measured.450 Absorbance value. Results are as follows: Figure 3 As shown, the recombinant nanobody VP3-Nb66 OD 450 =1.66, indicating that the recombinant nanobody VP3-Nb66 has good binding activity, which is the first report of its kind.
[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. However, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. A heavy chain antibody variable region sequence VP3-Nb66, characterized in that, Its amino acid sequence is SEQ ID NO:
2.
2. The heavy chain antibody variable region sequence VP3-Nb66 according to claim 1, characterized in that, The nucleotide sequence encoding the amino acid sequence of the variable region sequence VP3-Nb66 of the heavy chain antibody is SEQ ID NO:
1.
3. The screening method for a single heavy chain antibody variable region sequence VP3-Nb66 as described in claim 1, characterized in that, Includes the following steps: A computer-simulated molecular docking screening method was adopted. AlphaFold3 was used for high-precision 3D modeling of the antigen, RFdiffusion was used for antibody design backbone, and Proteinmpnn was used for sequence design. Using Muscovy duck parvovirus VP3 protein antigen as ligand, global docking calculations were performed. The optimal binding energy between the nanobody and the antigen was -319.33 kcal / mol, thus obtaining a heavy chain antibody variable region sequence VP3-Nb66.
4. A recombinant eukaryotic expression plasmid constructed using the variable region sequence VP3-Nb66 of a heavy chain antibody as described in claim 1.
5. The recombinant eukaryotic expression plasmid according to claim 4, characterized in that, The nucleotide sequence of the recombinant eukaryotic expression plasmid is SEQ ID NO:
3.
6. The method for constructing recombinant eukaryotic expression plasmids as described in claim 4, characterized in that, Includes the following steps: The heavy chain antibody variable region sequence VP3-Nb66 and the pPIC9K vector were digested with restriction endonucleases EcoRI and NotI, respectively, and then recovered by 1% agarose gel electrophoresis. The target fragment was ligated with T4 ligase and transformed into E. coli DH5α competent cells. The cells were plated and cultured overnight. The next day, a single colony was picked and sequenced. The correctly identified recombinant eukaryotic expression plasmid was named pPIC9K-DPV-VP3-Nb66.
7. A recombinant expression engineered strain constructed using the recombinant eukaryotic expression plasmid as described in claim 4.
8. The method for constructing the recombinant expression engineered strain as described in claim 6, characterized in that, Includes the following steps: The recombinant eukaryotic expression plasmid pPIC9K-DPV-VP3-Nb66 was linearized using the restriction endonuclease Sal I, added to Pichia pastoris X-33 competent cells, mixed well, and then transferred to a pre-chilled electroporation cuvette. After being placed on ice, the cuvette was transferred to an electroporator for electroporation. After electroporation, pre-chilled sorbitol was added, and the cuvette was pipetted and transferred to a centrifuge tube. The cuvette was incubated statically at 30°C, centrifuged at room temperature, and the cells were collected and resuspended in YPG medium. The cells were then spread onto YPG solid medium containing bleomycin and incubated at 37°C for 2-3 days. Single colonies were picked for PCR identification. If the identification was correct, the recombinant expression engineered strain was obtained.
9. A recombinant nanobody against Muscovy duck parvovirus VP3 protein prepared using the recombinant expression engineered strain described in claim 6.
10. The use of the recombinant nanobody against Muscovy duck parvovirus VP3 protein as described in claim 9 in the preparation of nanobody formulations for detecting Muscovy duck parvovirus disease.