PDCoV non-structural protein Nsp2 polyclonal antibody as well as preparation method and application thereof

Abstract

The invention provides a PDCoV non-structural protein Nsp2 polyclonal antibody and a preparation method and application thereof, and the method comprises the following steps: diluting purified recombinant Nsp2 protein with PBS to obtain a diluent; emulsifying the diluent and a freund's complete adjuvant according to a volume ratio of (0.5-1.5): 1 to obtain an emulsion; performing immunization on the rabbits according to the emulsion, wherein the immunization mode is subcutaneous injection; the emulsion is adopted for enhancing immunization of the rabbits twice every 10-18 days; and carrying out heart blood collection on the rabbit on the 7th-8th day after the two times of boosted immunization to obtain the polyclonal antibody. The polyclonal antibody prepared by the embodiment of the invention can specifically recognize the Nsp2 protein, and can be used for Nsp2 protein detection, protein function identification, cell sub-localization and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polyclonal antibody against porcine deltacoronavirus (PDCoV) nonstructural protein Nsp2, a preparation method thereof and an application thereof. Background Art

[0002] Porcine deltacoronavirus (PDCoV) is a pathogen of porcine intestinal diseases, which can infect pig herds of all ages, causing symptoms such as diarrhea, vomiting and dehydration similar to those of porcine epidemic diarrhea and transmissible gastroenteritis of pigs.

[0003] Nonstructural protein 2 (Nsp2) is a multi-domain and multifunctional protein, which varies greatly among different coronaviruses and there are few related studies. Nsp2 may have important biological functions in many aspects such as virus replication, regulation of host immune response, resistance to host antiviral mechanisms, and mutation and evolution. However, the research on the function of PDCoV Nsp2 is very limited. Summary of the Invention

[0004] The purpose of the present invention is to provide a polyclonal antibody against PDCoV nonstructural protein Nsp2, a preparation method thereof and an application thereof. The polyclonal antibody prepared by using Nsp2 has high detection sensitivity and specificity, can specifically recognize the Nsp2 protein, and can be used for the detection of the Nsp2 protein, the identification of its protein function, cell localization, etc., providing important experimental materials for studying the biological function of the Nsp2 protein.

[0005] In a first aspect, the present invention provides a preparation method of a polyclonal antibody against PDCoV nonstructural protein Nsp2, the method comprising:

[0006] Diluting the purified recombinant Nsp2 protein with PBS to obtain a dilution;

[0007] Emulsifying the dilution and Freund's complete adjuvant at a volume ratio of 0.5-1.5:1 to obtain an emulsion;

[0008] Immunizing rabbits according to the emulsion by subcutaneous injection;

[0009] Boosting the immunization of rabbits twice with the emulsion every 10-18 days;

[0010] Collecting blood from the hearts of rabbits on the 7th-8th day after boosting the immunization twice to obtain the polyclonal antibody.

[0011] Further, the concentration of the recombinant Nsp2 protein in the dilution is 0.3-0.7 g / L.

[0012] Further, the step of immunizing rabbits with the emulsion by subcutaneous injection includes:

[0013] The volume of the injected emulsion for each immunization or booster immunization is 180 - 220 μL, and the amount of the recombinant Nsp2 protein contained in the injected emulsion is 40 - 60 μg.

[0014] Further, the breed of the rabbits is New Zealand White rabbits.

[0015] In a second aspect, the present invention provides a polyclonal antibody against PDCoV non-structural protein Nsp2 prepared by the method for preparing a polyclonal antibody against PDCoV non-structural protein Nsp2 as described above.

[0016] In a third aspect, the present invention provides an application of the polyclonal antibody against PDCoV non-structural protein Nsp2 as described above in detecting the overexpression of pRK5-Flag-Nsp2 recombinant plasmid in Vero cells.

[0017] In a fourth aspect, the present invention provides an application of the polyclonal antibody against PDCoV non-structural protein Nsp2 as described above in detecting the overexpression of pRK5-Flag-Nsp2 recombinant plasmid in HEK 293T cells.

[0018] In a fifth aspect, the present invention provides an application of the polyclonal antibody against PDCoV non-structural protein Nsp2 as described above in detecting Nsp2 produced during the infection of LLc-PK1 cells with PDCoV at the same titer but different time points.

[0019] In a sixth aspect, the present invention provides an application of the polyclonal antibody against PDCoV non-structural protein Nsp2 as described above in detecting Nsp2 produced during the infection of LLc-PK1 cells with PDCoV at the same time point but different titers.

[0020] In a seventh aspect, the present invention provides an application of the polyclonal antibody against PDCoV non-structural protein Nsp2 as described above in detecting the cellular localization of Nsp2 protein.

[0021] Compared with the prior art, the embodiments of the present invention have the following advantages:

[0022] The polyclonal antibody prepared by the present invention has high sensitivity and specificity, can specifically recognize the Nsp2 protein, and can be used for the detection of the Nsp2 protein, the identification of its protein function, cellular sub-localization, etc., providing an important experimental tool for studying the biological function of the Nsp2 protein. Description of the Drawings

[0023] Figure 1 It is a diagram of the IFA result of transiently expressing Flag-Nsp2 in an embodiment of the present invention;

[0024] Figure 2 This is the Western Blot result diagram of transiently expressing Flag-Nsp2 in an embodiment of the present invention;

[0025] Figure 3 This is the Western Blot result diagram of PDCoV infecting LLC-PK1 cells at the same titer but different time points in an embodiment of the present invention;

[0026] Figure 4 This is the Western Blot result diagram of PDCoV infecting LLC-PK1 cells at the same time point but different titers in an embodiment of the present invention;

[0027] Figure 5 This is the laser confocal result diagram of the cellular localization of PDCoV-Nsp2 protein in an embodiment of the present invention.

[0028] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. Specific Embodiments

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art belonging to the field of the present invention. The words such as "including" used herein mean that the elements or items appearing before this word cover the elements or items listed after this word and their equivalents, without excluding other elements or items.

[0030] Example 1. Preparation of Polyclonal Antibody against Porcine Deltacoronavirus Nsp2

[0031] Dilute 50 μg of the purified recombinant Nsp2 protein with PBS to 100 μl to obtain a dilution solution, and the concentration of the recombinant Nsp2 protein in the dilution solution is 0.5 g / L;

[0032] The purified recombinant Nsp2 protein was emulsified with Freund's complete adjuvant at a volume ratio of 1:1 to obtain an emulsion. New Zealand white rabbits were immunized with the emulsion at an immunization dose of 50 μg of recombinant Nsp2 protein per rabbit and an injection volume of 200 μL, and the immunization method was subcutaneous injection. After that, the rabbits were injected with 200 μL of the emulsion every 14 days for booster immunization, and a total of 2 booster immunizations were performed. On the 7th day after the two booster immunizations, antiserum of the rabbits, that is, polyclonal antibody, was obtained by cardiac blood collection.

[0033] Example 2. Determination of antibody titer by indirect ELISA

[0034] The recombinant Nsp2 protein was diluted to 0.01 mg / mL with the coating solution; added to a 96-well reaction plate at 100 μL per well and incubated overnight at 4°C. The next day, the coating solution was poured off, and the plate was washed 3 times with PBST, 3 min each time, and finally patted dry on absorbent paper by inverting. 200 μL of 5% skim milk powder was added to each well and blocked at 37°C for 2 h, then washed 3 times with PBST, 3 min each time, and finally patted dry on absorbent paper by inverting. The polyclonal antibody prepared in Example 1 and skim milk powder were diluted at volume ratios of 1:4500, 1:13500, 1:40500, 1:121500, 1:364500, 1:1093500, 1:3280500, and 100 μL was added to each well of the 96-well plate. The rabbit serum before immunization was used as a negative control and incubated at 37°C for 1 h. After washing the plate, 100 μL of goat anti-rabbit IgG-HRP (diluted 1:10000) was added to each well and incubated at 37°C for 1 h. After washing the plate, TMB was added for color development reaction, and the absorbance at a wavelength of 450 nm was measured after terminating the reaction. The results of titer detection are shown in Table 1: The absorbance of the polyclonal antibody diluted to 3280500 times is more than twice the absorbance of the negative control, indicating that the titer of the rabbit anti-Nsp2 polyclonal antibody is as high as 10 6 。

[0035] Table 1

[0036]

[0037] Note: If the absorbance of the test specimen well is more than twice the absorbance of the negative serum, it can be judged as positive.

[0038] Example 3. Specificity analysis of polyclonal antibody I

[0039] To verify whether the obtained rabbit anti-Nsp2 polyclonal antibody can be used in the subsequent IFA experiment, the obtained polyclonal antibody was used to detect the overexpression of the pRK5-Flag-Nsp2 recombinant plasmid in Vero cells.

[0040] The specific operation steps of IFA are as follows:

[0041] (1) Plasmid transfection: Vero cells were pre-seeded in six-well plates. When the cell density reached 70%-80%, 2 μg of pRK5-Flag-Nsp2 recombinant plasmid was transfected into Vero cells in each well and cultured in an incubator for 24 h;

[0042] (2) Cell fixation: The medium of the above-infected Vero cells was discarded and washed twice with pre-cooled PBS buffer; 1 mL of 4% paraformaldehyde solution was added to each well to fix the cells at room temperature for 15 min and then washed three times with pre-cooled PBS buffer;

[0043] (3) Cell permeabilization: 1 mL of 0.25% Triton X-100 solution was added to each well of the above-fixed cells to permeabilize the cells at room temperature for 10 min and then washed three times with pre-cooled PBS buffer;

[0044] (4) Blocking: 1 mL of 3% bovine serum albumin (BSA) was added to each well of the fixed and permeabilized cells for blocking, incubated at 37 °C for 1 h, and then washed three times with pre-cooled PBS buffer;

[0045] (5) Primary antibody incubation: Mouse anti-Flag antibody (diluted 1:1000) and rabbit anti-Nsp2 polyclonal antibody (diluted 1:500) diluted with antibody diluent were added to the blocked cells, incubated at 37 °C for 2 h, and then washed three times with pre-cooled PBS buffer;

[0046] (6) Secondary antibody incubation: Fluorescent secondary antibody of the corresponding species was added to the cells after primary antibody incubation, incubated at 37 °C in the dark for 1 h, and then washed three times with pre-cooled PBS buffer;

[0047] (7) DAPI staining: 1 mL of diluted DAPI staining solution was added to each well of the above cells, incubated at 37 °C in the dark for 15 min, and then washed three times with pre-cooled PBS buffer;

[0048] (8) The prepared samples were observed using an inverted fluorescence microscope.

[0049] The IFA results are as Figure 1 shown. As Figure 1 can be seen, the mouse anti-Flag antibody can specifically detect the overexpressed Flag-Nsp2 protein with red fluorescence, and the rabbit anti-Nsp2 polyclonal antibody can specifically detect the overexpressed Flag-Nsp2 protein with green fluorescence. Both antibodies can specifically detect the overexpressed Nsp2 protein, and the two fluorescences fuse to present yellow, indicating co-localization of the two fluorescences. Therefore, this result shows that the polyclonal antibody prepared in the examples of the present invention can be used for IFA detection of transient expression of Nsp2 in cells.

[0050] Example 4. Specificity analysis of polyclonal antibody II

[0051] To verify whether the obtained polyclonal antibody against Nsp2 can be used in subsequent Western Blot experiments, the overexpression of the pRK5-Flag-Nsp2 recombinant plasmid in HEK 293T cells was detected using the obtained polyclonal antibody. The specific steps are as follows:

[0052] (1) Sample preparation: Mix the sample evenly with 5 μL of 5× Loading Buffer, denature at 100 °C for 15 min, then centrifuge at 12,000 rpm for 2 min for standby.

[0053] (2) Loading: Add the prepared sample into the sample wells of the SDS-PAGE gel, 10 μL per well. The loading volume of Protein Ladder is 7 μL.

[0054] (3) Electrophoresis: Add the protein electrophoresis buffer into the electrophoresis tank and the electrode tank. During gel running, first perform constant voltage electrophoresis at 80 V for 20 min, and then at 120 V for 1 h.

[0055] (4) Transfer: Select a 0.22 μm polyvinylidene fluoride (PVDF) membrane. Cut the membrane into an appropriate size as needed, mark it at the corner with a black oil-based pen, activate it in methanol for 30 s, and then equilibrate it in the transfer buffer. Take out the completed SDS-PAGE after electrophoresis, leaving only the separating gel part. Stack it up from bottom to top in the order of the transparent side of the clip, sponge, filter paper, PVDF membrane, separating gel, filter paper, sponge, and the black side of the clip. Exclude air bubbles with a plastic plate for each layer to form a "sandwich". Then place the made "sandwich" in the electrode tank for transfer, and then place the electrode tank in the electrophoresis tank (note the correct connection of the electrodes, black to black, red to red). Put an ice pack into the electrophoresis tank, pour the transfer buffer into the electrophoresis tank, and place the entire installed electrophoresis tank in a large ice box, and perform constant current electrophoresis at 200 mA for 80 min.

[0056] (5) Blocking: Place the transferred membrane in a box containing TBST, rinse it twice with TBST, and then add an appropriate amount of blocking solution. Place the box on a shaker and block at room temperature for 1 h.

[0057] (6) Incubate with primary antibody: Pour out the blocking solution, rinse the membrane three times with TBST. Add the mouse anti-Flag antibody diluted with antibody diluent (diluted 1:1000) and the rabbit anti-Nsp2 polyclonal antibody (diluted 1:500), and incubate overnight on a shaker at 4 °C.

[0058] (7) Incubate with secondary antibody: Add TBST to the box, wash it on a shaker for 10 min, and repeat the washing step three times. Add an appropriate amount of HRP-labeled goat anti-mouse secondary antibody diluted 1:5000 with the blocking solution, and incubate on a shaker at room temperature for 1.5 h.

[0059] (8) Exposure and development: Wash with TBST at room temperature on a shaker for 10 min and repeat 5 times. Use an electrochemiluminescence (ECL) developing solution. Take appropriate amounts of Solution A and Solution B and mix them in a 1:1 ratio in the dark. Drop the mixed solution evenly onto the membrane and shake the box to ensure that the surface of the membrane is fully contacted with the developing solution. Clamp the PVDF membrane out of the box and place it protein side down on the film scanner, taking care to remove air bubbles. Use Image Studio software for exposure, adjust the contrast, and save the picture.

[0060] The results of Western blot are as Figure 2 shown. The rabbit anti-Nsp2 polyclonal antibody can detect a band with a molecular weight consistent with the predicted nsp2 protein in the sample overexpressing nsp2, and the position of the band is the same as that detected by the anti-Flag monoclonal antibody. Therefore, this result indicates that the rabbit anti-Nsp2 polyclonal antibody prepared in the examples of the present invention can be used for Western Blot detection of transiently expressed Nsp2 in cells.

[0061] Example 5. Verification of LLC-PK1 cells infected with PDCoV at the same titer at different time points

[0062] Seed healthy LLC-PK1 cells into a 6-cm cell culture dish. After the cell monolayer covers the dish, incubate PDCoV with an MOI of 0.01 with the cells for 2 h, then change to DMEM medium and continue culturing. Collect cell samples at 12 h, 24 h, and 36 h after virus infection, and at the same time collect non-infected cells at 36 h as the control group. Detect the protein expression using the rabbit anti-Nsp2 polyclonal antibody, mouse anti-PDCoV S protein monoclonal antibody, and rabbit anti-β-actin antibody as primary antibodies (for the detailed detection process, refer to the specific steps of Western blot in Example 3). The detection results are as Figure 3 shown. The rabbit anti-Nsp2 polyclonal antibody can specifically detect a band of approximately 58 kDa after 24 h of virus infection. The size of this band is consistent with the prediction, and it increases with the prolongation of virus infection time, and there is no band at this position in the negative control. In addition, the detection samples were re-verified using the antibody against the PDCoV S protein, and a band of approximately 180 kDa was detected around 12 h after virus infection. The size of this band is consistent with the prediction, and it increases with the prolongation of virus infection time ( Figure 3 ), indicating that PDCoV successfully infects LLC-PK1 cells and proliferates normally. The results show that the prepared rabbit anti-Nsp2 polyclonal antibody can be used for detecting the Nsp2 protein of the virus during the virus infection process.

[0063] Example 6. Verification of LLC-PK1 cells infected with different titers of PDCoV at the same time point

[0064] Seed healthy LLC-PK1 cells into 6-cm cell culture dishes. When the cells grow to about 100%, incubate the cells with PDCoV at 10 -3 MOI, 10 -2 MOI, 10 -1 MOI and 1 MOI for 2 h, then change to DMEM medium and continue culturing. Collect cell samples 24 h after virus infection, and collect uninfected cells as a control group at the same time. Detect protein expression by using rabbit anti-Nsp2 polyclonal antibody, mouse anti-PDCoV S protein monoclonal antibody and rabbit anti-β-actin antibody as primary antibodies (for the detailed detection process, refer to the specific steps of Western blot in Example 3). The detection results are as shown in Figure 4 . The rabbit anti-Nsp2 polyclonal antibody can specifically detect a band of about 58 kDa in the samples infected with 10 - 1 MOI and 1 MOI of the virus. Compared with the inoculation group of 10 - 1 MOI, the band of about 58 kDa in the inoculation group of 1 MOI is significantly darker ( Figure 5 ). The results further indicate that the prepared rabbit anti-Nsp2 polyclonal antibody can successfully detect the expression of Nsp2 in PDCoV-infected samples, and the higher the MOI, the higher the expression level of the nsp2 protein.

[0065] Example 7. Detection of the cellular localization of Nsp2 protein by rabbit anti-Nsp2 polyclonal antibody

[0066] Seed healthy LLC-PK1 cells into confocal dishes. When the cells grow to about 100%, incubate the cells with 1 MOI of PDCoV for 2 h, then change to DMEM medium and continue culturing. Collect cell samples 24 h after virus infection. Detect the subcellular localization of Nsp2 protein by using rabbit anti-Nsp2 polyclonal antibody and mouse anti-PDCoV S protein monoclonal antibody as primary antibodies (for the detailed detection process, refer to the specific steps of IFA). Observe the cells under a confocal microscope. The observation results are as shown in Figure 5 . The rabbit anti-Nsp2 polyclonal antibody can specifically detect the Nsp2 protein of the virus, with green fluorescence. The mouse anti-PDCoV S antibody can specifically detect the S protein of the virus, with red fluorescence. The two fluorescences partially overlap and show yellow, and the two fluorescences exist in the same infected cell, indicating the co-localization of the S protein and the Nsp2 protein. The rabbit anti-Nsp2 polyclonal antibody can specifically detect the subcellular localization of the Nsp2 protein in virus-infected samples.

[0067] In summary, the polyclonal antibody prepared using Nsp2 in the embodiments of the present invention has high detection sensitivity and specificity, can specifically recognize the Nsp2 protein, and can be used for the detection of the Nsp2 protein, the identification of its protein function, cell localization, etc., providing important experimental materials for studying the biological function of the Nsp2 protein.

[0068] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are all within the scope and spirit of the present invention described in the claims. Moreover, the present invention described herein can have other embodiments and can be implemented or realized in various ways.

Claims

1. A method for preparing a polyclonal antibody against the non-structural protein Nsp2 of PDCoV, characterized in that, The method includes: Dilute the purified recombinant Nsp2 protein with PBS to obtain a dilution; Emulsify the dilution and Freund's complete adjuvant at a volume ratio of 0.5 - 1.5:1 to obtain an emulsion; Immunize rabbits with the emulsion by subcutaneous injection; Boost the immunity of rabbits with the emulsion twice every 10 - 18 days; Collect blood from the hearts of rabbits on the 7th - 8th day after two boosts to obtain polyclonal antibodies.

2. The preparation method of the PDCoV non-structural protein Nsp2 polyclonal antibody according to claim 1, characterized in that, The concentration of the recombinant Nsp2 protein in the dilution is 0.3 - 0.7 g / L.

3. The preparation method of the PDCoV non-structural protein Nsp2 polyclonal antibody according to claim 1, characterized in that, The step of immunizing rabbits with the emulsion by subcutaneous injection includes: The volume of the injected emulsion for each immunization or boost is 180 - 220 μL, and the amount of the recombinant Nsp2 protein contained in the injected emulsion is 40 - 60 μg.

4. The preparation method of the PDCoV non-structural protein Nsp2 polyclonal antibody according to claim 1, characterized in that, The breed of the rabbits is New Zealand white rabbits.

5. A polyclonal antibody against PDCoV non-structural protein Nsp2 prepared by the method for preparing a polyclonal antibody against PDCoV non-structural protein Nsp2 according to any one of claims 1 - 4.

6. An application of the polyclonal antibody against PDCoV non-structural protein Nsp2 according to claim 5 in detecting the overexpression of the pRK5-Flag-Nsp2 recombinant plasmid in Vero cells.

7. An application of the polyclonal antibody against PDCoV non-structural protein Nsp2 according to claim 5 in detecting the overexpression of the pRK5-Flag-Nsp2 recombinant plasmid in HEK 293T cells.

8. An application of the polyclonal antibody against PDCoV non-structural protein Nsp2 according to claim 5 in detecting Nsp2 produced during the infection of LLc-PK1 cells by PDCoV at the same titer but different time points.

9. An application of the polyclonal antibody against PDCoV non-structural protein Nsp2 according to claim 5 in detecting Nsp2 produced during the infection of LLc-PK1 cells by PDCoV at the same time point but different titers.

10. An application of the polyclonal antibody against PDCoV non-structural protein Nsp2 according to claim 5 in detecting the cellular localization of the Nsp2 protein.

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