Recombinant protein VP2-Cap as well as preparation method and vaccine thereof

Abstract

The invention relates to the technical field of biology, and discloses a recombinant protein VP2-Cap as well as a preparation method and a vaccine thereof. According to the present invention, the GoCV Cap gene segment and the GPV VP2 gene segment are successfully amplified, the fusion gene VP2-Cap segment is obtained, and the recombinant plasmid pET-28a-VP2-Cap is constructed and successfully expressed so as to obtain the recombinant protein VP2-Cap; the recombinant protein VP2-Cap is soluble and efficient in expression in an escherichia coli system, the problems of complex process and activity loss of inclusion body renaturation are avoided, and the recombinant protein VP2-Cap is high in activity and is an ideal antigen for preparing efficient subunit vaccines.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a recombinant protein VP2-Cap, its preparation method, and a vaccine. Background Technology

[0002] Goose circovirus (GoCV) and goose plague virus (GPV) are two important pathogens that threaten the goose farming industry. They often cause mixed infections in farming settings, resulting in significant economic losses to the goose farming industry.

[0003] Currently, there are no commercially available vaccines that can simultaneously prevent GoCV and GPV infections. Existing prevention and control measures mostly rely on the combined use of single-virus vaccines, which not only increases breeding costs and the complexity of immunization operations, but also has problems such as conflicting immunization procedures and incomplete immune protection, making it difficult to meet the disease prevention needs of large-scale breeding.

[0004] Cap protein is the main structural protein of GoCV, and VP2 protein is the core structural protein of GPV. Both have strong immunogenicity and are ideal targets for developing bivalent subunit vaccines of GoCV and GPV. If efficient fusion expression of the two proteins can be achieved, it is expected to obtain a fusion protein with dual immune protection, providing a core material basis for the development of bivalent vaccines.

[0005] However, existing technologies for preparing structural proteins using prokaryotic expression systems often face the following technical bottlenecks: First, Cap or VP2 proteins obtained by traditional prokaryotic expression methods often exist as insoluble inclusion bodies, requiring complex denaturation-renaturation processes. This is not only cumbersome but also results in low protein activity and poor recovery rates after renaturation. Second, both Cap and VP2 proteins are large viral structural proteins. When they are fused for expression, the large molecular weight and complex spatial structure of the fusion protein make it easy to form inclusion bodies, making it difficult to obtain soluble proteins with native conformations. Third, even after renaturation, proteins derived from inclusion bodies often fail to fold correctly and self-assemble into virus-like particles with native conformations, ultimately leading to a significant reduction in the immunogenicity of the protein.

[0006] Therefore, developing a method to efficiently obtain a soluble GoCV Cap / GPVVP2 fusion protein with native conformation and biological activity has become a pressing technical challenge in this field. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a recombinant protein VP2-Cap and its preparation method, which realizes its soluble and efficient expression in the Escherichia coli system and avoids the complex process of inclusion body refolding and the problem of activity loss.

[0008] The technical problem to be solved by the present invention is to provide a vaccine that contains key immunogens of GoCV and GPV and has excellent immunogenicity.

[0009] To address the aforementioned technical problems, the first aspect of this invention provides a recombinant protein VP2-Cap, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0010] Accordingly, the present invention also provides a DNA molecule encoding the recombinant protein VP2-Cap, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0011] Accordingly, the present invention also provides a method for preparing the recombinant protein VP2-Cap, comprising the following steps: (1) Amplify the GoCV Cap gene fragment and the GPV VP2 gene fragment; The nucleotide sequence of the upstream primer used to amplify the GoCV Cap gene fragment is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 4; the nucleotide sequence of the upstream primer used to amplify the GPV VP2 gene fragment is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 6. (2) Using the upstream primer with the nucleotide sequence shown in SEQ ID NO: 5 and the downstream primer with the nucleotide sequence shown in SEQ ID NO: 4, the obtained GoCV Cap gene fragment and GPV VP2 gene fragment were subjected to PCR amplification of the fusion gene fragment to obtain the VP2-Cap gene fragment. (3) The pMD-18T-VP2-Cap plasmid and the pET-28a(+) plasmid were double-digested and then ligated with the VP2-Cap gene fragment to construct the recombinant plasmid pET-28a-VP2-Cap; (4) The recombinant plasmid pET-28a-VP2-Cap was transformed into the expression strain, and the bacterial culture was obtained by expression; (5) The bacterial culture was purified to obtain the recombinant protein VP2-Cap.

[0012] As an improvement to the above scheme, in step (5), the recombinant plasmid pET-28a-VP2-Cap is transformed into the expression strain, and the bacterial culture is obtained by expression, including: The positive bacterial culture was inoculated into 100 mL of Kan LB broth and cultured with shaking until OD (Organic Degradation) was achieved. 600When the concentration reaches 0.6-0.7, IPTG is added to continue induction. After induction, centrifuge and discard the supernatant. Add PBS to the precipitate and sonicate on ice. After disruption, centrifuge again at 4°C and collect the disruption supernatant and disruption precipitate. Resuspend the disruption precipitate in an equal volume of PBS. The final concentration of IPTG is 0.75mM-1.2mM, the induction temperature is 14℃-18℃, and the induction time is 19h-21h.

[0013] As an improvement to the above scheme, step (5) involves purifying the bacterial culture, including: The soluble recombinant protein VP2-Cap in the lysate was captured and purified by nickel affinity chromatography under non-denaturing conditions to obtain high-purity recombinant protein VP2-Cap.

[0014] As an improvement to the above scheme, in step (1), PCR amplification is performed using pMD-18T-Cap and pMD-18T-VP2 cloning plasmids as templates to obtain the GoCV Cap gene fragment and the GPV VP2 gene fragment. The PCR amplification reaction system includes: 1 μL of GoCV Cap gene; 1 μL of GPV VP2 gene; 10 μL of 2×LA PCRMix; 1 μL of upstream primer; 1 μL of downstream primer; and 7 μL of ultrapure water. The PCR amplification reaction conditions included: pre-denaturation at 94℃-96℃ for 290s-310s; denaturation at 94℃-96℃ for 28s-32s, annealing at 56℃-60℃ for 28s-32s, extension at 70℃-74℃ for 78s-82s, for a total of 30 cycles; and a final extension at 70℃-74℃ for 590s-610s.

[0015] Accordingly, the present invention also provides a primer pair, including a first primer pair for amplifying the GoCV Cap gene fragment, a second primer pair for amplifying the GPV VP2 gene fragment, and a third primer pair for fusing amplification of the GoCV Cap gene fragment and the GPVVP2 gene fragment; The nucleotide sequence of the upstream primer of the first primer pair is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 4. The nucleotide sequence of the upstream primer of the second primer pair is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 6; The nucleotide sequence of the upstream primer of the third primer pair is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 4.

[0016] Accordingly, the present invention also provides a set of plasmids, including the pMD-18T-VP2-Cap plasmid and the recombinant plasmid pET-28a-VP2-Cap.

[0017] Accordingly, the present invention also provides a vaccine containing the recombinant protein VP2-Cap and an adjuvant.

[0018] Accordingly, the present invention also provides a method for preparing a vaccine, for preparing the VP2-Cap subunit vaccine, comprising: Dilute the recombinant protein VP2-Cap to a preset concentration; Mix and emulsify the adjuvant and diluted antigen.

[0019] Implementing this invention has the following beneficial effects: (1) In this invention, through a unique flexible linker peptide design and low-temperature induction process, the soluble and efficient expression of the large GoCV Cap and GPV VP2 fusion protein in the E. coli system is achieved, avoiding the complex process of inclusion body refolding and the problem of activity loss.

[0020] (2) In this invention, the recombinant protein VP2-Cap obtained contains the key immunogens of GoCV and GPV, and has excellent immunogenicity. It lays a solid material foundation for the development of a safe and efficient GoCV and GPV bivalent subunit vaccine, and achieves the goal of "one injection for multiple protections". At the same time, it can self-assemble into virus-like particles (VLPs), which maximizes the simulation of the conformation of natural viruses and has extremely strong immunogenicity. It is an ideal antigen for preparing efficient subunit vaccines.

[0021] (3) In this invention, the entire process is based on a mature Escherichia coli expression system, which is simple to operate, has good reproducibility, and is suitable for large-scale industrial production. Compared with the eukaryotic expression system, the cost is significantly reduced. Attached Figure Description

[0022] Figure 1 PCR results of the GoCV Cap gene fragment and GPV VP2 gene fragment in Example 1 of this invention, where M is DL2000 DNA Marker; 1 is GPV VP2 gene fragment; 2 is GoCV Cap gene fragment; and 3 is negative control. Figure 2: PCR results of the VP2-Cap gene fragment in Example 1 of the present invention, M is DL2000 DNA Marker; 1 is VP2-Cap fusion gene; 2 is negative control; Figure 3: PCR results of the recombinant cloning plasmid in Example 1 of the present invention; M: DL2000 plus DNA Marker; 1-7: Identification of recombinant cloning bacterial culture samples; 8: Negative control; Figure 4: Secondary structure analysis results of the VP2-Cap fusion protein in Example 1 of the present invention; Figure 5: Hydrophilicity and hydrophobicity analysis results of the VP2-Cap fusion protein in Example 1 of the present invention; Figure 6: Results of transmembrane structure analysis of the VP2-Cap fusion protein in Example 1 of this invention; Figure 7: Analysis results of the VP2-Cap fusion protein signal peptide in Example 1 of the present invention; Figure 8: Identification results of double enzyme digestion of recombinant plasmid pET-28a-VP2-Cap in Example 1 of this invention. M is DL2000plus Marker; 1 is pET-28a double enzyme digestion plasmid; 2 is pET-28a-VP2-Cap double enzyme digestion plasmid; 3 is VP2-Cap gene fragment. Figure 9: Expression results of recombinant protein VP2-Cap in Example 1 of the present invention; M is protein marker; 1 is pET-28a empty vector plasmid; 2 is fusion protein pET-28a-VP2-Cap prokaryotic plasmid; Figure 10: Purification and expression results of recombinant protein VP2-Cap in Example 1 of the present invention; In Figure 10A, M is the protein marker; 1-9 represent the protein elution solutions in sequence 1 to 9, respectively; In Figure 10B, M is the protein marker; 1 is the Western blot (WB) identification of the purified protein; In Figure 10C, M is the protein marker; 1 is the GoCV positive serum identification of recombinant protein VP2-Cap; In Figure 10D, M is the protein marker; 1 is the GPV positive serum identification of recombinant protein VP2-Cap. Figure 11 : Solubility analysis diagram of recombinant protein VP2-Cap in Example 2 of this invention; M is protein marker; 1 is the supernatant of pET-28a empty vector plasmid; 2 is the precipitate of pET-28a empty vector plasmid; 3 is the supernatant of recombinant protein VP2-Cap; 4 is the precipitate of recombinant protein VP2-Cap. Figure 12: Electrophoretic analysis diagram of the induced temperature optimization in Example 3 of the present invention; M is protein marker; 1 is the supernatant of recombinant protein VP2-Cap after 12℃ for 24 h; 2 is the precipitate of recombinant protein VP2-Cap after 12℃ for 24 h; 3 is the supernatant of recombinant protein VP2-Cap after 16℃ for 20 h; 4 is the precipitate of recombinant protein VP2-Cap after 16℃ for 20 h; 5 is the supernatant of recombinant protein VP2-Cap after 25℃ for 8 h; 6 is the precipitate of recombinant protein VP2-Cap after 25℃ for 8 h; 7 is the supernatant of recombinant protein VP2-Cap after 37℃ for 5 h; 8 is the precipitate of recombinant protein VP2-Cap after 37℃ for 5 h. Figure 13: Electrophoretic analysis of IPTG concentration optimization in Example 3 of the present invention; M is protein marker; 1 is whole bacteria before pET-28a-VP2-Cap induction; 2 is the supernatant after IPTG disruption at 1.25 mM concentration; 3 is the precipitate after IPTG disruption at 1.25 mM concentration; 4 is the supernatant after IPTG disruption at 1 mM concentration; 5 is the precipitate after IPTG disruption at 1 mM concentration; 6 is the supernatant after IPTG disruption at 0.75 mM concentration; 7 is the precipitate after IPTG disruption at 0.75 mM concentration. Figure 14: Electron micrograph of recombinant protein VP2-Cap. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the present invention clearer, specific embodiments will be described in further detail below.

[0024] The sources of the materials involved in the various embodiments of the present invention are as follows: The prokaryotic expression vector pET-28a(+) was purchased from Ubisoft Biotechnology Co., Ltd.; HindIII, Xho I restriction endonucleases, T4 ligase, 10×T4 DNA Ligase buffer, LA Taq enzyme, and BL21(DE3) competent cells were all purchased from BioNTech Biotechnology (Beijing) Co., Ltd.; pMD-18 was purchased from Takara; Code No. 6011 (pMD TM18-T Vector Cloning Kit); Gel extraction kit purchased from Omega; Code No. D2500-01 (Gel Extraction Kit D2500); Plasmid extraction kit purchased from Tiangen Biotech (Beijing) Co., Ltd.; Kanamycin sulfate and methanol purchased from Guangzhou Yongjin Biotechnology Co., Ltd.; BCA protein assay kit, IPTG, Ni-Agarose His-tagged protein purification kit, HisTag mouse monoclonal antibody, HRP-labeled goat anti-mouse IgG, 5×SDS gel loading buffer, ultrasensitive ECL chemiluminescence kit, SDS-PAGE gel preparation kit, and skim milk powder purchased from Beyotime Biotechnology Co., Ltd.

[0025] Example 1 Recombinant protein VP2-Cap and its preparation method 1. Primer design and synthesis for amplifying the GoCV Cap gene fragment and the GPV VP2 gene fragment: The stop and start codons of the GPV VP2 and GoCV Cap genes were removed, respectively. During vector construction, a HindIII restriction site and the Kozak expression regulatory element (ACCACC) sequence were introduced to the 5' end of the upstream primer of GPV VP2. An XhoI restriction site was added to the 5' end of the downstream primer of GoCV Cap. CCC and CCG protective bases were introduced upstream of each restriction site to ensure restriction efficiency. A gene expressing the flexible linker peptide (G4S2) was introduced to the 3' end of the GPV VP2 gene, and a reverse complementary gene expressing G4S2 was introduced to the 5' end of the GoCV Cap gene. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The nucleotide sequences of the synthesized primers are shown in SEQ ID NO: 3-6, as detailed in Table 1 below. Table 1 Primer Sequences

[0026] It should be noted that in Table 1 above, the underlined lines represent restriction enzyme sites, and the wavy lines represent Kozak sequences.

[0027] 2. PCR amplification of the GoCV Cap gene fragment and the GPV VP2 gene fragment: Using pMD-18T-Cap and pMD-18T-VP2 cloning plasmids as templates, 20 μL PCR amplification reaction systems were established. The GoCV Cap gene fragment and GPV VP2 gene fragment were amplified sequentially using the primers in Table 1. The amplification systems are shown in Table 2. Table 2. Amplification systems of GoCV Cap gene fragment and GPV VP2 gene fragment

[0028] The PCR amplification reaction conditions included: 95℃ pre-denaturation for 300 s; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 80 s, for a total of 30 cycles; and a final extension at 72℃ for 600 s. The PCR products were analyzed and identified by 1.2% agarose gel electrophoresis. Figure 1 ), yielding specific bands of approximately 786 bp and 1784 bp.

[0029] It should be noted that the pMD-18T vector is a commercially available linear vector, and the pMD-18T-Cap and pMD-18T-VP2 cloning plasmids were constructed using the TA cloning method. Specifically, the construction methods for the pMD-18T-Cap and pMD-18T-VP2 cloning plasmids include the following steps: (1) Amplification of the target fragment Viral DNA was extracted from tissues of geese infected with GoCV and GPV, respectively, and then the target fragments of Cap and VP2 were amplified by PCR using primers in Table 3.

[0030] The PCR amplification reaction conditions included: 95℃ pre-denaturation for 300 s; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 80 s, for a total of 30 cycles; and a final extension at 72℃ for 600 s. The PCR products were analyzed and identified by 1.2% agarose gel electrophoresis, yielding specific bands of approximately 753 bp and 1761 bp.

[0031] Table 3 Primer Sequences

[0032] (2) Recovery and purification of the target fragment The Cap and VP2 gene fragments obtained from the above gel electrophoresis were excised under UV light, and the target bands were transferred to clean 2mL centrifuge tubes and weighed. 0.1 times the weight of the gel block was added to sol-gel buffer XP2, and the mixture was incubated in a 50℃ water bath for 6-10 minutes, gently inverting the centrifuge tube repeatedly until the gel block was completely dissolved. After the solution cooled to room temperature, it was transferred to a DNA purification column, centrifuged at 10000×g for 1 minute, and the eluent was discarded. 350μL of binding buffer was added to the column, centrifuged at 12000×g for 1 minute, and the waste liquid was discarded. 650μL of wash buffer SPW was added, centrifuged for 1 minute under the same conditions, and the waste liquid was discarded. This washing process was repeated once. The empty column was centrifuged at 12000×g for 2 minutes to completely remove residual ethanol, and allowed to stand at room temperature for 5 minutes to allow residual solvent to evaporate. Finally, add 20 μL of elution buffer EB to the center of the column membrane, let it stand at room temperature for 2 min, centrifuge at 13000×g for 1 min to collect the DNA elution buffer, and store it at -20℃ for later use.

[0033] (3) Ligation and transformation of the Cap gene Following the operating instructions of the pMD-18T vector cloning kit, the DNA fragments purified by gel electrophoresis were ligated with the linearized vector. The specific reaction system was prepared according to Table 4. After vortexing to mix all components, the reaction solution was collected by momentary centrifugation in a microcentrifuge and then placed at a constant temperature of 16°C for the ligation reaction.

[0034] Table 4 Connection Reaction System

[0035] After ligation, the ligation products pMD-18T-Cap and pMD-18T-VP2 plasmids were obtained. An appropriate amount of the ligation products was used for transformation: DH5α competent cells were removed from -80℃, thawed on ice, and then all the ligation solution was added. The mixture was gently mixed and incubated on ice for 30 min. Subsequently, the cells were heat-shocked at 42℃ for 90 s, and immediately returned to ice for 2 min. 500 μL of sterile LB liquid medium was added, and the cells were incubated at 37℃ with shaking at 200 rpm for 45 min. The cells were centrifuged at 12000×g for 5 min, and a portion of the supernatant was discarded. Approximately 180 μL of the bacterial culture was retained to resuspend the precipitate, which was then evenly spread onto LB agar plates containing ampicillin (100 μg / mL) and incubated upside down at 37℃ for 16–18 h.

[0036] (3) Identification of pMD-18T-Cap and pMD-18T-VP2 gene recombinant plasmids Single colonies were picked and cultured in LB (Amp+) liquid medium at 37°C with shaking for 12 hours. Using the cultured bacterial solution as a template, the single-clone bacterial solution was identified according to the following amplification reaction and system. The positive bacterial solutions were sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing.

[0037] The PCR amplification reaction conditions included: 95℃ pre-denaturation for 300s; 95℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 80s, for a total of 30 cycles; and a final extension at 72℃ for 600s.

[0038] The recombinant cloning bacterial culture was identified by PCR using the M13 primers in Table 5, and target bands of 908bp and 1916bp were obtained, indicating that the recombinant cloning plasmids pMD-18T-Cap and pMD-18T-VP2 were successfully constructed.

[0039] Table 5 M13 primer sequences

[0040] 3. Gene fusion PCR amplification of GoCV Cap gene fragment and GPV VP2 gene fragment Using the electrophoretically purified GoCV Cap gene fragment and GPV VP2 gene fragment as templates, the fusion gene fragment was amplified in vitro using GPV VP2-F (SEQ ID NO: 5) primers and GoCV Cap-R primers (SEQ ID NO: 4). The specific amplification system is shown in Table 6. Table 6 Gene Fusion PCR Amplification System

[0041] The PCR amplification reaction conditions included: 95℃ pre-denaturation for 600 s; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 80 s, for a total of 32 cycles; and a final extension at 72℃ for 600 s. The PCR product was analyzed and identified by 1.2% agarose gel electrophoresis (Figure 2), yielding a VP2-Cap gene fragment of approximately 2540 bp.

[0042] 4. Cloning of the VP2-Cap gene fragment (1) Recovery and purification of the target fragment The VP2-Cap gene fragment obtained by PCR amplification was subjected to 1.2% agarose gel electrophoresis. The target band was excised under UV light, transferred to a clean 2mL centrifuge tube, and weighed. Subsequently, the target fragment was recovered and purified according to step (2) of the above-mentioned method for constructing pMD-18T-Cap and pMD-18T-VP2 cloning plasmids.

[0043] (2) Ligation and transformation of the VP2-Cap gene In this step, the ligation process is performed according to the method in step (3) of the construction method of pMD-18T-Cap and pMD-18T-VP2 cloning plasmids described above, specifically the ligation and transformation of the Cap gene. After ligation, the ligation product pMD-18T-VP2-Cap plasmid is obtained. Subsequently, the ligation product is transformed according to the method in step (3) of the construction method of pMD-18T-Cap and pMD-18T-VP2 cloning plasmids described above.

[0044] (3) Identification of VP2-Cap gene recombinant plasmid Single colonies were picked and cultured in LB (Amp+) liquid medium at 37°C with shaking for 12 hours. Using the cultured bacterial solution as a template, the single-clone bacterial solution was identified according to the following amplification reaction and system. The positive bacterial solutions were sent to Beijing Qingke Biotechnology Co., Ltd. for Sanger sequencing.

[0045] The PCR amplification reaction conditions included: 95℃ pre-denaturation for 600s; 95℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 80s, for a total of 32 cycles; and a final extension at 72℃ for 600s.

[0046] The recombinant cloning bacterial culture was identified by PCR using the M13 primers in Table 5 (Figure 3), and a target band of 2689 bp was obtained, indicating that the recombinant cloning plasmid was successfully constructed.

[0047] 5. Biological information analysis of the VP2-Cap fusion protein (1) The primary structure of the fusion protein was predicted using the software DNAStar and ProtParam; The primary structure of the VP2-Cap fusion protein was predicted using the ProtParam analysis tool. The results showed that the VP2-Cap fusion protein consists of 847 amino acid residues, with a molecular weight of approximately 110 kDa. The molecular formula of the protein is C2. 3934 H 6354 N 1202 O 1200 S 39 .

[0048] (2) Use Protean software to predict the secondary structure of the fusion protein; The secondary structure of the VP2-Cap fusion protein was predicted and analyzed using Protean and SOPMA, and the results are shown in Figure 4. Figure 4 reveals that the fusion protein contains 5.43% α-helix structure, 21.96% β-sheet structure, and 72.61% random coil region. Epitope analysis showed potential antigenic determinants at amino acid positions 1-50, 200-250, 300-350, 400-500, 550-650, and 700-750, with the C-terminal region exhibiting significantly higher antigenic accessibility than other regions.

[0049] (3) Use the online software ProtScale to analyze the hydrophilicity and hydrophobicity of proteins; The hydrophilicity and hydrophobicity of the VP2-Cap fusion protein were analyzed using the Protscale algorithm, and the results are shown in Figure 5. Figure 5 shows that the hydrophobicity index of this protein is mainly distributed in the negative region, indicating that it has obvious hydrophilic characteristics and can be classified as a hydrophilic protein.

[0050] (4) Use the online server TMHMM to predict transmembrane domains; The VP2-Cap fusion protein was analyzed using the TMHMM 2.0 transmembrane structure prediction algorithm, and the results are shown in Figure 6. The results indicate that no transmembrane domains were detected in the protein sequence. Based on its characteristics, it is speculated that this protein may be a secretory protein.

[0051] (5) Predict signal peptides using the online software SignalP 4.1; Signal peptide analysis of the VP2-Cap fusion protein was performed using the SignalP 4.1 server, as shown in Figure 7. The prediction results indicate that the protein sequence does not contain a typical signal peptide domain.

[0052] 6. Construct the recombinant plasmid pET-28a-VP2-Cap pMD-18T-VP2-Cap plasmid and pET-28a(+) plasmid were extracted and double-digested overnight at 37℃. The system is shown in Table 7 below.

[0053] Table 7 Double enzyme digestion system

[0054] The enzyme digestion products were purified by 1.2% agarose gel electrophoresis. Subsequently, the vector and the VP2-Cap gene fragment were ligated at 16℃ for 4 h to construct the recombinant plasmid pET-28a-VP2-Cap (nucleotide sequence shown in SEQ ID NO: 2). The ligation reaction system is shown in Table 8 below.

[0055] Table 8 Connection Reaction System

[0056] The recombinant plasmid was digested using a HindIII and XhoI double enzyme digestion system for verification. 1% gel electrophoresis analysis (Figure 8) confirmed that the VP2-Cap fusion gene fragment had been correctly inserted into the vector.

[0057] 7. Transformation and identification of recombinant plasmid pET-28a-VP2-Cap DH5α competent cells, frozen at -80℃, were rapidly transferred to ice for thawing. 10 μL of the ligation product was gently mixed with the cell suspension and incubated on ice for 30 min. Subsequently, a heat shock treatment was performed at 42℃ for 90 s, followed immediately by cooling on ice for 5 min. 900 μL of sterile LB liquid medium was added, and the mixture was placed in a 37℃ shaker at 200 rpm for 45 min. The transformed bacterial culture was evenly spread onto LB agar plates supplemented with kanamycin (50 μg / mL) and incubated at 37℃ for 12–16 h. Seven morphologically uniform single colonies were then selected and inoculated into LB liquid medium containing kanamycin, and cultured at 37℃ with a shaker (200 rpm) for 4 h. Positive clones were screened by PCR using the culture as a template. The positive bacterial culture was expanded and cultured overnight. The recombinant plasmid was extracted according to the instructions of the plasmid extraction kit. After double enzyme digestion at 37°C, the plasmid was identified by agarose gel electrophoresis and sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0058] 8. Expression of recombinant protein VP2-Cap The correctly sequenced recombinant plasmid pET28a-VP2-Cap was transformed into BL21(DE3) competent cells. After culturing for 14-16 h, a single colony was picked and inoculated into 1 mL of LB (Kan+) liquid medium. The cells were incubated at 37°C with shaking for 4 h. Positive bacterial cultures were identified using the T7 primers in Table 9. 1 mL of the positive bacterial culture was inoculated into 100 mL of Kan LB broth containing 50 μg / mL. The cells were incubated at 37°C with shaking until the OD600 reached 0.6. Then, 1 mM IPTG was added, mixed, and induced for another 20 h at 16°C and 200 rpm. After centrifugation at 10000×g for 5 min, the supernatant was discarded, and PBS was added to the precipitate for sonication on ice. After disruption, the cells were centrifuged at 4°C and 12000×g for 10 min. The supernatant and precipitate (amino acid sequence as shown in SEQ ID NO: 1) were collected, and the precipitate was resuspended in an equal volume of PBS.

[0059] Table 9 T7 Primer Sequences

[0060] The collected fragmented supernatant and fragmented precipitate were subjected to SDS-PAGE gel electrophoresis. Western blotting analysis of the recombinant protein was performed using mouse anti-His antibody as the primary antibody, and the results are shown in Figure 9. The results showed that the recombinant protein was detectable in both the fragmented supernatant and the fragmented precipitate, exhibiting a specific band in the relative molecular weight range of approximately 100-135 kDa, with a size of 110 kDa, consistent with the expected target band, indicating successful expression of the fusion protein.

[0061] 9. Purification of recombinant protein VP2-Cap The recombinant fusion protein was purified according to the instructions of the His-tagged protein purification kit (denaturing formulation). The specific steps are as follows: (1) After the recombinant protein VP2-Cap was expressed, 800 mL of the induced bacterial culture was collected, and the bacterial pellet was obtained by centrifugation at 12000×g for 5 min. The bacterial pellet was then resuspended in 20 mL of non-denaturing lysis buffer.

[0062] (2) Place the bacterial suspension on ice and sonicate it for 5 seconds, with a 5-second interval, for a total of 15 minutes.

[0063] (3) Collect the broken suspension, centrifuge at 12000×g for 30 min at 4℃, and collect the broken supernatant.

[0064] (4) Add 16 mL of gel to a 50 mL EP tube, centrifuge at 1000 × g for 1 min at 4 °C, discard the supernatant, add an equal amount of non-denaturing lysis buffer to equilibrate the gel, and repeat the equilibration twice.

[0065] (5) Add the broken supernatant after centrifugation to the equilibrated gel, mix thoroughly, and incubate at 4°C and 45 r / min on a water shaker for 3 h.

[0066] (6) Add the mixture to the affinity chromatography column and let it stand until a gel precipitate appears.

[0067] (7) Open the stopcock at the bottom of the chromatography column and wash the column 5 times with 2 column volumes of non-denaturing lysis buffer, 1 mL each time.

[0068] (8) Wash the column 10 times with non-denaturing washing solution, 1 mL each time.

[0069] (9) Elute the target protein with non-denaturing elution buffer, 1 mL each time, collect the eluted liquid each time, and measure the protein content using a micro spectrophotometer until no protein can be detected, then stop elution.

[0070] (10) Wash the gel with 5 mL of non-denaturing lysis buffer 2-3 times, soak it in 20% anhydrous ethanol, and store it at 4°C.

[0071] Each eluted fraction was collected separately, and protein purity was analyzed by SDS-PAGE electrophoresis. The results are shown in Figure 10. A relatively single recombinant protein eluate was obtained on the 9th elution (Figure 10A). Western blotting of the purified protein eluate yielded a single band (Figure 10D), indicating successful purification of the recombinant VP2-Cap protein. Further verification of the fusion protein's biological activity was conducted by reacting GoCV and GPV-positive serum with the recombinant protein. The results showed that both GoCV and GPV-positive serum specifically reacted with the recombinant protein (Figures 10B and 10C).

[0072] Example 2 Solubility analysis of recombinant protein VP2-Cap Prepare the protein gel for SDS-PAGE electrophoresis according to the SDS-PAGE gel preparation kit instructions. Take 16 μL of sample and 4 μL of 5× protein loading buffer, mix thoroughly by pipetting, and place in a boiling water bath for 10 min. Load 10 μL of sample onto the gel and perform polyphenylene gel electrophoresis at 120V. Perform solubility analysis of the target protein using Western blotting, as follows: (1) Cut a PVDF membrane of appropriate size and place it in a methanol solution to activate for 60 seconds, then immediately transfer it to the transfer solution.

[0073] (2) After the sample is subjected to SDS-PAGE gel electrophoresis, the stacking gel on the top layer of the protein gel and the bromophenol blue layer on the bottom layer are removed.

[0074] (3) Using the semi-dry transfer method, filter paper, protein gel and filter paper are stacked in sequence on the transfer apparatus, the voltage is adjusted to 25V, 0.5A, and constant rotation is performed for 15min.

[0075] (4) After the transfer, place the PVDF membrane in a clean box containing 5% skim milk powder and seal it.

[0076] (5) After sealing at 4℃ overnight, wash the membrane 5 times with TBST solution for 5 minutes each time.

[0077] (6) Dilute the primary antibody (His Tag Mouse Monoclonal Antibody) with TBST solution at a ratio of 1:2500, mix thoroughly, incubate at room temperature for 1.5 h, and then wash the membrane 5 times with TBST solution for 5 min each time.

[0078] (7) Dilute the secondary antibody (HRP-labeled goat anti-mouse IgG) with TBST solution at a ratio of 1:8000, mix thoroughly, incubate at room temperature for 1 h, and then wash the membrane 5 times with TBST solution for 5 min each time.

[0079] (8) Add the color developing solution droplets onto the PVDF membrane and place it in the developing analyzer for developing and imaging.

[0080] The results are as follows Figure 11 As shown in the analysis, the recombinant protein exists in the form of a soluble protein.

[0081] Example 3 Optimization of expression conditions for recombinant protein VP2-Cap (1) Optimal induction temperature optimization Take 100 μL of the preserved bacterial culture and inoculate it into 10 mL of LB (Kan+) liquid medium. Incubate at 37 °C and 200 rpm for 18 hours. Then, inoculate 200 μL of the overnight culture into 20 mL of LB (Kan+) liquid medium and continue to incubate at 37 °C and 200 rpm until the OD600 reaches 0.6. Add 1 mM IPTG to the final concentration. The cells were cultured at 12℃ for 24 h at 200 rpm; 16℃ for 20 h at 200 rpm; 25℃ for 8 h at 200 rpm; and 37℃ for 5 h at 200 rpm. After centrifugation at 8000×g for 5 min, the cells were washed three times with PBS buffer, resuspended in PBS, and sonicated on ice. Then, the cells were centrifuged at 12000×g for 15 min at 4℃. The supernatant and precipitate were collected, and the collected cell suspension was sonicated on ice at 200W for 5 s, with 5 s intervals, for a total of 15 min. 20 μL of each supernatant and precipitate was mixed with 5 μL of 5×SDS-PAGE loading buffer, boiled for 10 min, and then analyzed by SDS-PAGE electrophoresis.

[0082] The results are shown in Figure 12. The fusion protein was present in the supernatant and precipitate when the induction temperature and time were 12℃ for 24h and 16℃ for 20h. The expression level in the supernatant was higher at 16℃ for 20h, while the recombinant protein was present in the form of precipitate at 25℃ for 8h and 37℃ for 5h.

[0083] (2) Optimal IPTG concentration optimization 100 μL of the preserved bacterial culture was added to 10 mL of LB (Kan+) liquid medium and incubated at 200 r / min at 37 °C for 16 h. 200 μL of the overnight culture was added to 20 mL of LB (Kan+) liquid medium and incubated at 200 r / min at 37 °C until the OD600 reached 0.6. IPTG was then added to final concentrations of 0.75 mM, 1 mM, and 1.25 mM, respectively, and incubated at 16 °C for 18 h. After centrifugation at 8000 × g for 5 min, the bacterial cells were collected, washed three times with PBS buffer, resuspended in PBS, and sonicated on ice. The cells were then centrifuged at 12000 × g for 15 min at 4 °C, and the supernatant and precipitate were collected. 20 μL of each supernatant and precipitate was mixed with 5 μL of 5 × SDS-PAGE loading buffer, boiled, and analyzed by SDS-PAGE electrophoresis.

[0084] As shown in Figure 13, the expression level of the fusion protein was the highest when the final IPTG concentration was 1 mM, indicating that the optimal IPTG concentration was 1 mM.

[0085] Example 4: Determination of Recombinant Protein VP2-Cap Concentration Following the operating manual of the BCA protein quantification kit provided by Beyotime Biotechnology Co., Ltd., the concentration of the purified fusion protein sample was quantitatively analyzed. The specific steps are as follows: (1) Take 20 μL of 25 mg / mL protein standard and prepare 0.5 mg / mL protein standard in 980 μL of PBS buffer. Mix thoroughly and set aside.

[0086] (2) Prepare BCA working solution by mixing reagent A and reagent B in a volume ratio of 50:1, mix thoroughly, and use immediately after preparation.

[0087] (3) Use PBS buffer to fill each reaction well to a final volume of 20 μL. After thorough mixing, prepare standard solutions with a concentration gradient of 0-0.5 mg / mL, with specific concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg / mL.

[0088] (5) Add 200 μL of pre-prepared BCA working solution to each well, gently shake to mix, avoid generating bubbles, and incubate at 37°C in the dark for 30 min.

[0089] (6) Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at a wavelength of 562 nm.

[0090] (7) Calculate the protein concentration of the sample to be tested by plotting the standard curve based on the standard.

[0091] The recombinant protein was purified and concentrated using the BCA protein concentration assay kit, and the protein concentration was measured to be 1.215 mg / mL.

[0092] Example 5 Electron Microscopy Identification of Recombinant Protein VP2-Cap Take an appropriate amount of purified protein, fix it with glutaraldehyde overnight and place it at 4℃. Take 20 μL of the overnight treated protein and drop it onto a 200-mesh copper grid. Aspirate the excess liquid and let it air dry at room temperature. Negative stain with 2% phosphotungstic acid solution for 5 min. Aspirate the excess liquid and let it air dry. Observe the morphology of the sample using transmission electron microscopy.

[0093] As shown in Figure 14, spherical particles of about 20nm-25nm can be observed, with morphology similar to natural GoCV and GPV virus particles, indicating that the purified recombinant protein can be assembled into virus-like particles.

[0094] The above description is merely a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. Therefore, any equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A recombinant protein VP2-Cap, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A DNA molecule encoding the recombinant protein VP2-Cap as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

2.

3. A method for preparing the recombinant protein VP2-Cap as described in claim 1, characterized in that, Includes the following steps: (1) Amplify the GoCV Cap gene fragment and the GPV VP2 gene fragment; The nucleotide sequence of the upstream primer used to amplify the GoCV Cap gene fragment is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 4; the nucleotide sequence of the upstream primer used to amplify the GPV VP2 gene fragment is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

6. (2) Using the upstream primer with the nucleotide sequence shown in SEQ ID NO: 5 and the downstream primer with the nucleotide sequence shown in SEQ ID NO: 4, the obtained GoCV Cap gene fragment and GPV VP2 gene fragment were subjected to PCR amplification of the fusion gene fragment to obtain the VP2-Cap gene fragment. (3) The pMD-18T-VP2-Cap plasmid and the pET-28a(+) plasmid were double-digested and then ligated with the VP2-Cap gene fragment to construct the recombinant plasmid pET-28a-VP2-Cap; (4) The recombinant plasmid pET-28a-VP2-Cap was transformed into the expression strain, and the bacterial culture was obtained by expression; (5) The bacterial culture was purified to obtain the recombinant protein VP2-Cap.

4. The method for preparing the recombinant protein VP2-Cap as described in claim 3, characterized in that, In step (5), the recombinant plasmid pET-28a-VP2-Cap is transformed into the expression strain, and the bacterial culture is obtained by expression, including: The positive bacterial culture was inoculated into 100 mL of Kan LB broth and cultured with shaking until OD (Organic Degradation) was achieved. 600 When the concentration reaches 0.6-0.7, IPTG is added to continue induction. After induction, centrifuge and discard the supernatant. Add PBS to the precipitate and sonicate on ice. After disruption, centrifuge again at 4°C and collect the disruption supernatant and disruption precipitate. Resuspend the disruption precipitate in an equal volume of PBS. The final concentration of IPTG is 0.75mM-1.2mM, the induction temperature is 14℃-18℃, and the induction time is 19h-21h.

5. The method for preparing the recombinant protein VP2-Cap as described in claim 2, characterized in that, In step (5), the bacterial culture is purified, including: The soluble recombinant protein VP2-Cap in the lysate was captured and purified by nickel affinity chromatography under non-denaturing conditions to obtain high-purity recombinant protein VP2-Cap.

6. The method for preparing the recombinant protein VP2-Cap as described in claim 2, characterized in that, In step (1), PCR amplification was performed using pMD-18T-Cap and pMD-18T-VP2 cloning plasmids as templates to obtain the GoCV Cap gene fragment and the GPVVP2 gene fragment. The PCR amplification reaction system includes: 1 μL of GoCV Cap gene; 1 μL of GPV VP2 gene; 10 μL of 2×LA PCR Mix; 1 μL of upstream primer; 1 μL of downstream primer; and 7 μL of ultrapure water. The PCR amplification reaction conditions included: pre-denaturation at 94℃-96℃ for 290s-310s; denaturation at 94℃-96℃ for 28s-32s, annealing at 56℃-60℃ for 28s-32s, extension at 70℃-74℃ for 78s-82s, for a total of 30 cycles; and a final extension at 70℃-74℃ for 590s-610s.

7. Primer combination, characterized in that, It includes a first primer pair for amplifying the GoCV Cap gene fragment, a second primer pair for amplifying the GPV VP2 gene fragment, and a third primer pair for fusing and amplifying the GoCV Cap gene fragment and the GPV VP2 gene fragment; The nucleotide sequence of the upstream primer of the first primer pair is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

4. The nucleotide sequence of the upstream primer of the second primer pair is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 6; The nucleotide sequence of the upstream primer of the third primer pair is shown in SEQ ID NO: 5, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

4.

8. A set of plasmids, characterized in that, Includes the pMD-18T-VP2-Cap plasmid as described in claim 3 and the recombinant plasmid pET-28a-VP2-Cap.

9. A vaccine, characterized in that, The vaccine contains the recombinant protein VP2-Cap as described in claim 1 and an adjuvant.

10. A method for preparing a vaccine, used to prepare the VP2-Cap subunit vaccine as described in claim 9, characterized in that, include: Dilute the recombinant protein VP2-Cap according to claim 1 to a predetermined concentration; Mix and emulsify the adjuvant and diluted antigen.

Images