Coxsackie virus B3 3C protease recombinant protein, polyclonal antibody and preparation method and application thereof

Abstract

This invention discloses a recombinant protein of Coxsackievirus B3 type 3C protease, a polyclonal antibody, and their preparation method and applications. Based on the CVB3 genome sequence, full-length primers were designed, and the 3C fragment was obtained by PCR amplification. The pET28a-3C recombinant expression vector was constructed, transformed into competent bacteria, and induced to express by IPTG. The CVB3 3C protease recombinant protein was then purified. Using this recombinant protein as an immunogen, it was emulsified with Freund's adjuvant and used to immunize animals. After multiple immunizations, serum was collected to obtain the CVB3 3C protein polyclonal antibody. The prepared polyclonal antibody exhibits high specificity, specifically recognizing the 3C protein band at 28 kDa. It can be widely applied in detection methods such as Western blot, immunofluorescence, and immunoprecipitation for the expression analysis, intracellular localization study, and screening of interacting proteins of the 3C protein during CVB3 infection.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a recombinant protein of Coxsackievirus B3 type 3C protease, a polyclonal antibody, and their preparation methods and applications. Background Technology

[0002] Coxsackievirus B3 (CVB3) belongs to the Enterovirus genus of the Picornaviridae family. Infection with CVB3 can cause a variety of diseases, including viral myocarditis, meningitis, pancreatitis, and hand-foot-and-mouth disease. Currently, CVB3 is considered the most likely pathogen to cause viral myocarditis, associated with 20%–25% of myocarditis cases. Among CVB3-infected individuals, approximately 10%–20% of cases can progress to dilated cardiomyopathy, heart failure, or even sudden cardiac death, posing a serious threat to human health and becoming a global public health concern.

[0003] CVB3 is a non-enveloped, single-stranded, positive-sense RNA virus with a genome size of approximately 7.4 kb. Its structure is 5'-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3', which translates into a structural protein precursor P1 and non-structural protein precursors P2 and P3, each consisting of 2193 amino acids. Non-structural proteins are fundamental to viral replication within host cells. Among them, non-structural protein 3C is an important protease of Coxsackievirus. Besides cleaving its own proteins, it can also regulate viral replication by cleaving or directly binding to host proteins, suggesting that 3C plays a crucial role in CVB3 pathogenesis. 3C has a unique amino acid sequence and spatial conformation, is expressed only in CVB3-infected cells, and is produced in large quantities in the early stages of infection. It can serve as a specific biomarker for early CVB3 infection, viral replication activity, and disease progression.

[0004] Currently, methods for in vivo detection of CVB3 are mainly limited to its structural protein VP1. This structural protein shares high homology with other enteroviruses, easily leading to cross-reactivity and false positives. However, non-structural proteins, as important proteases of CVB3, can better reflect the dynamic replication process of CVB3 within host cells. Existing research on the CVB3 3C protein focuses primarily on its function in cleaving other proteins, while other mechanisms of action of 3C remain unclear. Crucially, as a key protease of CVB3, there are currently no commercially available polyclonal antibodies for 3C, severely hindering research on the functions of the 3C protein and the development of CVB3 detection technologies.

[0005] Therefore, developing a polyclonal antibody that can specifically recognize the CVB3 non-structural protein 3C is of great clinical value and application prospect for filling the gap in the detection of CVB3 and 3C proteins and promoting related basic research and clinical testing applications. Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant protein of Coxsackievirus B3 type 3C protease and its preparation method.

[0007] Another objective of this invention is to provide a polyclonal antibody against Coxsackievirus B3 type 3C protein prepared by immunization with the above-mentioned recombinant protein and a method thereof.

[0008] Another object of the present invention is to provide the use of the above-mentioned polyclonal antibody in the preparation of reagents or kits for detecting Coxsackievirus B3 type 3C protein.

[0009] Another object of the present invention is to provide a detection kit for Coxsackievirus B3 type 3C protein containing the above-mentioned polyclonal antibody.

[0010] To achieve the above-mentioned objective, this invention provides a method for preparing recombinant Coxsackievirus B3 type 3C protease protein, comprising the following steps: (1) Based on the full-length 3C sequence of the CVB3 genome in NCBI GenBank: M33854.1 as shown in SEQ ID NO: 1, full-length primers were designed: the upstream primer sequence is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO: 4; (2) Using the full-length 3C sequence shown in SEQ ID NO: 1 as a template, PCR amplification was performed using the full-length primers described in step (1) to obtain the 3C fragment; (3) The 3C fragment obtained in step (2) and the pET28a plasmid were double-digested, and the digested products were recovered after separation by agarose gel electrophoresis. (4) The enzyme digestion product recovered in step (3) is subjected to T4 ligation reaction, and the ligation product is transformed into competent bacteria; (5) The transformed competent bacteria were added to induce expression with IPTG. The bacterial cells were then disrupted by sonication and treated with NTA-Ni. 2+ Column affinity purification was performed, and the elution buffer was collected to obtain the recombinant protein of Coxsackievirus B3 type 3C protease.

[0011] Preferably, the induction conditions in step (5) are: final IPTG concentration of 0.8 mM, temperature of 28℃, rotation speed of 160 rpm, and induction for 8-12 h.

[0012] To achieve the above-mentioned objective, the present invention also provides a recombinant Coxsackievirus B3 type 3C protease protein obtained by the preparation method described in claim 1 or claim 2, the amino acid sequence of which is shown in SEQ ID NO:2.

[0013] To achieve the above-mentioned objectives, this invention also provides a method for preparing a polyclonal antibody against Coxsackievirus B3 type 3C protein. Using the recombinant protein as an immunogen, the recombinant protein is mixed with Freund's complete adjuvant or Freund's incomplete adjuvant at a 1:1 ratio and then thoroughly ground until the protein is completely emulsified. Animals are immunized on days 1, 14, 21, 28, and 35, respectively. Blood is collected 7 days after the last immunization, and serum is separated to obtain the polyclonal antibody against Coxsackievirus B3 type 3C protein.

[0014] To achieve the above-mentioned objectives, the present invention also provides a polyclonal antibody against Coxsackievirus B3 type 3C protein obtained by the above preparation method.

[0015] To achieve the above-mentioned objectives, the present invention also provides the application of the above-mentioned polyclonal antibody in the preparation of reagents or kits for detecting Coxsackievirus B3 type 3C protein.

[0016] Furthermore, the detection is a Western blot assay, and the working dilution ratio of the polyclonal antibody is 1:(2000-3000).

[0017] Furthermore, the detection is an immunofluorescence assay used to detect the intracellular expression and localization of Coxsackievirus B3 type 3C protein, and the working dilution ratio of the polyclonal antibody is 1:500.

[0018] Furthermore, the detection is an immunohistochemical assay used to detect the expression and localization of Coxsackievirus B3 type 3C protein in tissues, and the working dilution ratio of the polyclonal antibody is 1:100.

[0019] Furthermore, the detection is an immunoprecipitation assay used to detect the interacting proteins of Coxsackievirus B3 type 3C protein, and the working amount of the polyclonal antibody is 1.0-4.0 μg antibody / mg total protein.

[0020] To achieve the above-mentioned objectives, the present invention also provides a Coxsackievirus B3 type 3C protein detection kit, which contains the above-mentioned polyclonal antibody.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a new strategy for using CVB3 non-structural protein 3C as the detection target; non-structural protein 3C has good conservation and low cross-reactivity, which significantly improves the specificity and accuracy of detection.

[0022] (2) The CVB3 3C protein polyclonal antibody prepared by the present invention fills the gap in the current lack of CVB3 in vitro replication detection antibodies; the antibody can not only be used to detect viral replication and intracellular localization, but also to analyze interacting proteins, breaking through the limitation of existing technologies that can only rely on a single indicator VP1.

[0023] (3) The antibody of the present invention can be applied to various detection methods such as Western blot, immunofluorescence, immunohistochemistry, and immunoprecipitation, and can be further developed into a detection kit, providing a new and reliable tool for basic research and clinical detection of CVB3, with a wide range of applications. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the double enzyme digestion verification results of the pET28a-3c recombinant plasmid in Example 1, where: lane M is the 2000 DNA Marker; lanes 1 and 2 are the double enzyme digestion results of the pET28a-3c recombinant plasmid; lane 3 is the double enzyme digestion result of the pET28a vector; Figure 2 This is a Coomassie Brilliant Blue staining image of the target band in Example 1, where: lane M is the protein marker; lane 3C is the induction of Rosetta bacteria by transformation of the recombinant pET28a-3c plasmid; Figure 3 The His tag of purified protein 3C was detected by Western blot in Example 1, wherein: lane 1 is after the unmodified pET28a plasmid was transformed into Rosetta bacteria and induced; lane 2 is after the pET28a-3c recombinant plasmid was transformed into Rosetta bacteria and induced. Figure 4 This is a schematic diagram of the ELISA detection results of the 3C polyclonal antibody titer in Example 3; Figure 5 This is a schematic diagram of the Western blot detection results for the recognition of CVB3 3C protein by 3C in Example 4; Figure 6 This is a schematic diagram of the results of immunofluorescence detection of CVB3 3C protein in mouse heart tissue in Example 4; Figure 7 This is a schematic diagram of the immunofluorescence detection results of CVB3 3C protein in HeLa cells in Example 4; Figure 8 This is a schematic diagram showing the results of immunohistochemical detection of CVB3 3C protein in mouse heart tissue in Example 4; Figure 9 This is a schematic diagram of the results of immunoprecipitation analysis of 3C protein-interacting proteins in Example 4. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0026] HeLa cells were purchased from Fenghui Biotechnology Co., Ltd. HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum. Example 1

[0027] A method for preparing a recombinant protein of Coxsackievirus B3 type 3C protease includes the following steps: (1) Full-length primer design: The full-length 3C sequence of CVB3 (GenBank: M33854.1) was searched in NCBI (as shown in SEQ ID NO:1), and the full-length sequence was synthesized at Sangon Biotech (Shanghai) Co., Ltd. Based on the above sequence, full-length primers were designed, and the upstream and downstream primer sequences are as follows: Upstream primer (SEQ ID NO:3): 5'-CCGCTCGAGGGCCCTGCCTTTGAGTTCGC-3' (underlined is the XhoI restriction site); Downstream primer (SEQ ID NO:4): 5'-CCGGAATTCTTGCTCATCATTGAAGTAGT-3' (underlined is the EcoRI restriction site); product size is 549 bp.

[0028] (2) Obtaining the 3C fragment: Using the full-length 3C sequence shown in SEQ ID NO: 1 as a template, PCR amplification was performed using the specific primers described above. The amplification system was as follows: 10 μL TaKaRa Taq™ Version, 0.5 μL each of forward and reverse primers (20 mM), 1 μL cDNA template, and 8 μL nuclease-free water. Amplification program: 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 60 s, for 30 cycles; 72℃ for 7 min. The 3C fragment was recovered using a product recovery kit.

[0029] (3) Place the 3C fragment and pET28a plasmid obtained in step (2) in a 37℃ metal bath for double digestion, and add 10× loading buffer to terminate the digestion reaction. Separate the target band by agarose gel electrophoresis with an agar concentration of 1.5%, and recover the fragment from the agar.

[0030] (4) T4 ligation of 3C fragment: The enzyme digestion product recovered in step (3) is subjected to T4 ligation reaction to ligate the 3C fragment to pET28a plasmid, and the resulting ligation product is introduced into Rosetta competent cells.

[0031] The results were verified by double enzyme digestion of the recombinant pET28a plasmid, as follows: Figure 1 As shown, there is a specific 3C gene band at around 500 bp and a specific pET28a plasmid band at around 3000 bp.

[0032] (5) The transformed competent bacteria were added to induce expression with IPTG. The bacterial cells were then disrupted by sonication and treated with NTA-Ni. 2+ Column affinity purification followed by collection of elution buffer yields the recombinant Coxsackievirus B3 type 3C protease protein; the specific process is as follows: (5-1) High expression of 3C recombinant protein; ① Three bacterial strains were selected as experimental subjects: empty Rosetta bacteria (without any plasmid transformation), Rosetta bacteria containing an empty vector (negative control), and Rosetta bacteria containing a 3C recombinant vector (positive recombinant bacteria). The overnight culture was transferred to LB broth containing kanamycin at a ratio of 1:100 and cultured at 37°C and 250 rpm on a shaker until OD600 ≈ 0.6. IPTG was added to a final concentration of 0.8 mM, and the culture was induced at 28°C and 160 rpm for 8-12 h.

[0033] ② After cultivation, collect the bacterial culture, centrifuge at 13,000 rpm for 1 min, discard the supernatant, add 5 mL of buffer to the centrifuge tube, and sonicate (sonication time 10 s, interval 10 s, for 30 min, amplitude 3, operation on ice). Centrifuge the sonicated solution at 10,000 rpm, 4℃ for 60 min, transfer the supernatant to a plastic centrifuge tube, and add 1 mL of NTA-Ni. 2+ The column was placed on ice and mixed on a shaker for 1 h. The mixture was then transferred to a 5 mL syringe, and wash buffer was added to remove contaminating proteins. When the wash buffer oozes out at OD... 280 When the concentration is < 0.01, add elution buffer to the syringe and collect the buffer. Analyze the purity of the purified protein using SDS-PAGE. Figure 2 The results showed a distinct and specific target band around 28 kDa.

[0034] (5-2) Concentration and purification of 3C recombinant protein; The supernatant containing 3C protein was filtered through a 0.45 μm filter to remove impurities. The Ni-NTA affinity chromatography column was equilibrated with a buffer containing 20 mM TrisHCl and 0.5 M NaCl (pH 8.0) and pretreated at a flow rate of 0.8 mL / min. The filtered protein sample was loaded onto the column at the same flow rate. After the 3C protein specifically bound to nickel ions, a gradient of imidazole buffers (20 mM, 60 mM, 200 mM, 500 mM) was used for stepwise elution. Each eluent was collected until the G250 assay solution no longer showed color. After elution, the column was rinsed with three column volumes of deionized water, and the column was blocked with 20% ethanol. The collected eluent fractions at various concentrations were analyzed by SDS-PAGE electrophoresis to screen for high-purity target protein fractions. These fractions were then dialyzed into PBS buffer and stored at -80°C for later use. The results of protein purity and His tag verification are attached. Figure 3 .

[0035] The recombinant Coxsackievirus B3 type 3C protease protein prepared in this embodiment has the amino acid sequence shown in SEQ ID NO:2. Example 2

[0036] A method for preparing a polyclonal antibody against the Coxsackievirus B3 type 3C protein includes the following steps: Before each immunization, the recombinant protein prepared in Example 1 and Freund's incomplete adjuvant or Freund's complete adjuvant are mixed in a mortar at a ratio of 1:1 and then ground thoroughly until the protein and adjuvant are completely mixed and form a uniform milky white viscous consistency, so that the protein is completely emulsified.

[0037] Two New Zealand White rabbits, aged 8-9 weeks and weighing approximately 2.5 kg, were selected and acclimatized in an animal laboratory for one week. Immunization was then performed according to the steps in Table 1. Table 1 Animal Immunization Procedures Seven days after the last immunization, lidocaine was injected into the marginal ear vein of rabbits until the rabbit muscles were completely relaxed. A 60 mL sterile syringe was then inserted vertically into the heart to collect blood. The collected blood was placed in a sterile 50 mL centrifuge tube, incubated overnight at 4°C, and centrifuged at 4000 rpm for 10 min. The serum was then collected in a sterile centrifuge tube and stored at -20°C or -80°C to obtain the CVB3 3C protein polyclonal antibody. Example 3

[0038] The titer of the CVB3 3C protein polyclonal antibody prepared in Example 2 was detected by ELISA. The specific steps are as follows: ① Dilute the 3C protein to 1.0 μg / mL, use PBS buffer as the coating solution, add 100 μL of the coated protein to a 96-well plate, and incubate overnight at 4°C. The next day, wash three times with PBST buffer, add 200 μL of 5% BSA, block at 37°C for 1 h, and wash three more times with PBST buffer.

[0039] ② Negative controls (using pre-immunization serum as a negative control) and post-immunization serum were serially diluted at ratios of 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, and 1:64000. 100 μL of the diluted sample was added to each pre-coated 96-well plate, with three replicates per sample. The plates were incubated at 37°C for 1 h. After washing three times with PBST buffer, 200 μL of HRP-labeled goat anti-rabbit IgG was added to each well to label the primary antibody (i.e., 3C polyclonal antibody) that binds to the antigen. The plates were incubated at 37°C for 40 min. After washing three times with PBST buffer and twice with ultrapure water, 100 μL of TMB chromogenic solution (Beyotime, P0209) was added. The plates were incubated at room temperature in the dark for 20 min, and the reaction was terminated by adding 100 μL of hydrochloric acid.

[0040] ③ Use an ELISA reader to measure the absorbance (OD) of each well at 450 nm. 450 (Value). Potency is defined as antiserum OD. 450 Value / Pre-immune serum OD 450 The highest dilution factor with a value > 2.1. Results showed that 3C polyclonal serum remained positive at a dilution ratio of 1:32000, which is the highest dilution factor for detection (see attached). Figure 4 ). Example 4

[0041] Specific detection of anti-rabbit 3C polyclonal antibody for recognizing 3C protein in tissues (1) Western blot detection ① Sample preparation: Take approximately 100 mg of CVB3-infected tissue (or CVB3-infected in vitro cultured cells), homogenize the tissue, add 500 μL of protein lysis buffer (Kangwei Century, CW2333S), and lyse on ice for 40 min, vortexing once every 10 min. Centrifuge at 12,000 rpm, 4℃ for 30 min, collect the supernatant, quantify BCA protein, add 5× Loading buffer, denature at 100℃ for 10 min, and store at -20℃ for later use.

[0042] ② SDS-PAGE electrophoresis and transfer: After separation by 12% SDS-PAGE electrophoresis, the protein samples were transferred to a methanol-activated PVDF membrane for 10 min (BioRad, 10026938). After the transfer, the PVDF membrane was immediately blocked with 5% BSA in a shaker at room temperature for 2 h.

[0043] ③ Antibody incubation and chemiluminescence: The membrane was immersed in the working solution of the anti-rabbit 3C protein polyclonal antibody primary antibody prepared in Example 2 (3C polyclonal antibody diluted 1:2000 in 5% BSA) and incubated overnight at 4°C. After discarding the primary antibody, the membrane was washed four times with PBST for 10 min each time. HRP-labeled secondary antibody (Jackson, AB_2313567) was added, and the membrane was incubated on a shaker at room temperature for 1 h. The membrane was then washed four times with PBST for 10 min each time. The membrane was developed using ECL chemiluminescence. The results showed that the 3C polyclonal antibody could specifically detect the expression of the 3C protein of CVB3 (see attached image). Figure 5 ).

[0044] (2) Immunofluorescence of tissues and organs ① Tissue sample preparation: Fresh tissue specimens from CVB3-infected mice were fixed in 4% paraformaldehyde for at least 24 hours. After washing with PBS, the specimens were immediately placed in OCT embedding medium (SAKURA, 4583) and then rapidly frozen in liquid nitrogen. The frozen tissue blocks were fixed to the sample head of a cryostat, equilibrated at -25°C, and sections of approximately 8 μm thickness were continuously cut. After air-drying at room temperature, the sections were used for subsequent staining or stored in a sealed container at -80°C.

[0045] ② Staining and Result Interpretation: After thawing at room temperature for approximately 30 min, sections were permeabilized with 0.1% Triton X-100 for 30 min and washed three times with PBS. Sections were blocked with 5% BSA for 30 min, then diluted 3C polyclonal antibody (3C polyclonal antibody diluted 1:500 in 5% BSA) was added, and the sections were incubated overnight at 4°C in a humidified chamber. The next day, after washing three times with PBS, FITC-labeled fluorescent secondary antibody (Invitrogen, A-11008) was added, and the sections were incubated at 37°C in the dark for 1 h. After discarding the fluorescent secondary antibody, sections were washed three times with PBS, and DAPI working solution was added. The sections were stained at room temperature in the dark for 10 min. After discarding the DAPI working solution, sections were washed three times with PBS, and an anti-fluorescence quencher (brand name) was added to the sections. The tissue was covered with a coverslip, and the 3C polyclonal antibody detection was observed using a confocal microscope. During result interpretation, DAPI showed blue cell nuclei, and the green fluorescent signal represented 3C protein. Different intensities of green fluorescence were observed based on the expression level of 3C protein (see attached image). Figure 6 ).

[0046] (3) Immunofluorescence of cultured cells ① Preparation of in vitro cultured cell samples: CVB3-infected or 3C plasmid-transfected cells were added to cell slides and cultured for 12 h. The culture medium was discarded, the cells were washed three times with PBS, and fixed with 4% paraformaldehyde for at least 30 min.

[0047] ② Staining and Result Interpretation: Discard 4% paraformaldehyde and wash three times with PBS. Permeabilize with 0.1% Triton X-100 for 30 min, then wash three times with PBS. Block sections with 5% BSA for 30 min, then add diluted 3C polyclonal antibody (3C polyclonal antibody diluted 1:500 in 5% BSA) and incubate overnight at 4°C in a humidified chamber. The next day, wash three times with PBS, then add FITC-labeled fluorescent secondary antibody (Invitrogen, A-11008) and incubate at 37°C in the dark for 1 h. Discard the fluorescent secondary antibody, wash three times with PBS, and use an anti-fluorescence quencher as a mounting medium. Place the cell slides on a glass slide and observe the 3C polyclonal antibody detection under a confocal microscope. During result interpretation, DAPI showed blue cell nuclei, and the green fluorescent signal represented 3C protein. Different intensities of green fluorescence were observed according to the expression levels of 3C protein (see attached). Figure 7 ).

[0048] (4) Immunohistochemistry of tissues and organs ① Tissue sample preparation: Fresh tissue specimens from CVB3-infected mice were fixed in 4% paraformaldehyde for at least 24 hours, embedded in paraffin, and sectioned.

[0049] ② Staining and Result Interpretation: First, paraffin sections were baked at 65℃, dehydrated, and antigen-retrieved, then blocked with 5% BSA for 2 h. A 1:100 dilution of 3C protein polyclonal antibody was added, and the sections were incubated overnight at 4℃. The next day, after washing with PBS, HRP-labeled secondary antibody was added, and the sections were incubated at room temperature for 1 h. DAB staining was performed (a suitable amount of reddish-brown color was observed under the microscope), and the staining was stopped by placing the sections in double-distilled water. Hematoxylin staining of the nuclei was performed for 10 min. After dehydration with ethanol and xylene, the sections were mounted with neutral resin. Final interpretation: Cell nuclei appeared blue (hematoxylin staining signal), and positive cells appeared brownish-yellow (DAB staining signal). See Appendix. Figure 8 .

[0050] (5) Immunoprecipitation ① Sample preparation: After intracellular infection with CVB3, add an appropriate amount of protein lysis buffer (Kangwei Century, CW2334S), place on ice for lysis for 40 min, vortex once every 10 min. Centrifuge at 12000 rpm, 4℃ for 30 min, and collect the supernatant. Quantify BCA protein, which is the total cellular protein.

[0051] ② Immunoprecipitation: Total cellular protein was divided into three groups: Input, IgG, and IP, and treated as follows. Input was directly added to 5× Loading buffer, denatured at 100℃ for 10 min, and stored at -20℃ for later use. For each 1.0 mg protein sample, 2 μg of 3C polyclonal antibody was added, and the sample was incubated overnight at 4℃. For each 1.0 mg protein sample, 2 μg of rabbit IgG antibody (Proteintech, 30000-0-AP) was added, and the sample was incubated overnight at 4℃. An appropriate amount of A / G magnetic beads was placed in a magnetic rack, the buffer solution in the beads was discarded, and the beads were washed three times with an appropriate amount of PBS. The washed magnetic beads were added to the overnight IgG and IP samples and incubated at room temperature for 4 h. The IgG and IP samples incubated with the magnetic beads were placed in a magnetic rack, and the supernatant was discarded. The magnetic beads were gently washed three times with an appropriate amount of PBS, then 1× Loading buffer diluted with pure water was added, denatured at 100℃ for 10 min, and stored at -20℃ for later use.

[0052] ③ For Western blot detection: After separation by 10% SDS-PAGE, protein samples were transferred to a methanol-activated PVDF membrane. Transfer was performed for 30 min, followed immediately by blocking with 5% BSA on a shaker at room temperature for 2 h. The membrane was then immersed in a primary antibody working solution prepared with a protein antibody that interacts with the 3C protein (the concentration of the primary antibody working solution was determined by the antibody against the target interacting protein) and incubated overnight at 4°C. After discarding the primary antibody, the membrane was washed four times with PBST for 10 min each time. HRP-labeled secondary antibody (Jackson, AB_2313567) was added, and the membrane was incubated on a shaker at room temperature for 1 h. The membrane was then washed four more times with PBST for 10 min each time. Developed using ECL chemiluminescence. For result interpretation, Input was the positive control, IgG was the negative control, and IP was the experimental group (see attached diagram). Figure 9 ).

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A process for the preparation of recombinant protein of Coxsackievirus B3 3C protease, characterized by, Includes the following steps: (1) Based on the full-length 3C sequence of the CVB3 genome in NCBI GenBank: M33854.1 as shown in SEQ ID NO: 1, full-length primers were designed: the upstream primer sequence is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO: 4; (2) Using the full-length 3C sequence shown in SEQ ID NO: 1 as a template, PCR amplification was performed using the full-length primers described in step (1) to obtain the 3C fragment; (3) The 3C fragment obtained in step (2) and the pET28a plasmid were double-digested, and the digested products were recovered after separation by agarose gel electrophoresis. (4) The enzyme digestion product recovered in step (3) is subjected to T4 ligation reaction, and the ligation product is transformed into competent bacteria; (5) The transformed competent bacteria are added to IPTG to induce expression, and the bacteria are broken by ultrasonic wave, and then NTA-Ni 2+ column affinity purification, and the elution buffer is collected to obtain the recombinant protein of coxsackievirus B3 3C protease.

2. The method for preparing recombinant Coxsackievirus B3 type 3C protease protein according to claim 1, characterized in that, The induction conditions in step (5) are: final IPTG concentration of 0.8 mM, temperature of 28℃, rotation speed of 160 rpm, and induction for 8-12 h.

3. A recombinant protein of Coxsackievirus B3 type 3C protease obtained by the preparation method of claim 1 or claim 2, the amino acid sequence of which is shown in SEQ ID NO:

2.

4. A method for preparing a polyclonal antibody against the Coxsackievirus B3 type 3C protein, characterized in that, Using the recombinant protein of claim 3 as an immunogen, the recombinant protein is mixed with Freund's complete adjuvant or Freund's incomplete adjuvant at a ratio of 1:1 and then thoroughly ground until the protein is completely emulsified. Animals are immunized on days 1, 14, 21, 28 and 35, respectively. Blood is collected 7 days after the last immunization, and serum is separated to obtain the polyclonal antibody against Coxsackievirus B3 type 3C protein.

5. A polyclonal antibody against Coxsackievirus B3 type 3C protein obtained by the preparation method described in claim 4.

6. The use of the polyclonal antibody according to claim 5 in the preparation of reagents or kits for detecting Coxsackievirus B3 type 3C protein.

7. The application according to claim 6, characterized in that, The detection method is Western blot detection, and the working dilution ratio of the polyclonal antibody is 1:(2000-3000).

8. The application according to claim 6, characterized in that, The detection method is an immunofluorescence assay used to detect the intracellular expression and localization of Coxsackievirus B3 type 3C protein, and the working dilution ratio of the polyclonal antibody is 1:500; the detection method is an immunohistochemical assay used to detect the tissue expression and localization of Coxsackievirus B3 type 3C protein, and the working dilution ratio of the polyclonal antibody is 1:

100.

9. The application according to claim 6, characterized in that, The detection method is an immunoprecipitation assay used to detect the interacting proteins of Coxsackievirus B3 type 3C protein. The working amount of the polyclonal antibody is 1.0-4.0 μg antibody / mg total protein.

10. A Coxsackievirus B3 type 3C protein detection kit, characterized in that, It includes the polyclonal antibody as described in claim 5.

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