Sigma NS polyclonal antibody preparation method

Abstract

The invention relates to the technical field of sigma NS protein, and particularly discloses a preparation method of a sigma NS polyclonal antibody, which comprises the following steps: construction of pET-28a-S3 plasmid, expression and purification of His tag recombinant protein, and preparation and identification of the polyclonal antibody. The present invention provides an efficient method for extracting and purifying [sigma] NS protein from nelson bay positive reovirus (NBV). A pET-28a-S3 recombinant plasmid is constructed and is efficiently expressed and purified in escherichia coli, so that high-purity sigma NS recombinant protein with a His tag is obtained and is used as an ideal antigen for preparing a polyclonal antibody. According to the method, the problems of low antigen purity, poor antibody specificity and the like in the traditional antibody preparation process are effectively solved. Meanwhile, the identification result shows that the antibody has high specific binding capacity to sigma NS protein and low cross reaction with other related proteins, the quality and usability of the antibody are ensured, and the antibody has wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of σNS protein, and particularly relates to a method for preparing a σNS polyclonal antibody. Background Art

[0002] In biological and medical research, polyclonal antibodies are widely used because of their relatively simple preparation, low cost and ability to recognize multiple antigen epitopes.

[0003] As an important component of Nelson Bay Orthoreovirus (NBV), σNS protein plays a key role in viral replication and pathogenesis. Therefore, the preparation of polyclonal antibodies against σNS protein is of great significance for in-depth study of the biological characteristics of NBV and the development of diagnostic reagents and vaccines.

[0004] However, traditional polyclonal antibody preparation methods often have problems such as low antigen purity and poor antibody specificity, which affect the quality and application effect of the antibody. In particular, due to the complexity of NBV and the particularity of σNS protein, an efficient and reliable antibody preparation method is needed.

[0005] The present invention aims to provide a new method for preparing σNS polyclonal antibodies. Summary of the invention

[0006] The object of the present invention is to provide a method for preparing a σNS polyclonal antibody to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing a σNS polyclonal antibody comprises the following steps:

[0009] Construction of S1 and pET-28a-S3 plasmids

[0010] S11. Viral RNA extraction: RNA was extracted from Nelson Bay Orthoreovirus (NBV) and purified by TRIzol lysis, chloroform extraction, and isopropanol precipitation.

[0011] S12, reverse transcription of RNA into cDNA: reverse transcription of RNA into cDNA using reverse transcriptase, first remove genomic DNA, then perform reverse transcription reaction;

[0012] S13, target gene amplification: primers were designed according to the NBV-S3 sequence in GenBank, and PCR amplification was performed using cDNA as a template to obtain the target gene S3 fragment, gene S3 is shown in SEQ ID NO: 1;

[0013] S14, plasmid construction: The target gene S3 and the expression vector pET-28a were double-digested, the digested products were purified and then connected to obtain the recombinant plasmid pET-28a-S3, the recombinant plasmid was transformed into BL21 competent cells, the positive clones were screened, and the plasmid was extracted and sequenced for verification;

[0014] S2. Expression and purification of His-tagged recombinant proteins

[0015] S21. Small-scale induced expression: The E. coli transformed with the recombinant plasmid was induced to express at different temperatures and IPTG concentrations, and the expression products were analyzed by SDS-PAGE electrophoresis;

[0016] S22, large-scale induction expression: select the optimal expression conditions for large-scale induction, collect the bacteria and lyse them to obtain a lysate containing the target protein;

[0017] S23, purification: Purify the target protein using a gravity column, and obtain a highly pure His-tagged σNS recombinant protein through equilibration, loading, washing and elution steps;

[0018] S24, dialysis and concentration: The purified protein is dialyzed to remove small molecule impurities and concentrated using PEG12000 to obtain a high concentration of the target protein.

[0019] S3. Preparation and identification of polyclonal antibodies

[0020] S31. Immunization of animals: Use purified σNS recombinant protein as antigen to immunize rabbits for the first time, and perform booster immunization regularly;

[0021] S32. Collect serum: One week after the fourth immunization, collect rabbit serum, centrifuge to remove impurities and store at -80°C;

[0022] S33. Antibody titer detection: The titer of antibodies in serum was detected by ELISA method to determine the activity of antibodies;

[0023] S34. Identification of antibody specificity.

[0024] Preferably, the specific method for extracting viral RNA in S11 is to take out the Nelson Bay Orthoreovirus (NBV) virus frozen at -80°C, thaw it at room temperature, place it in a 1.5mL centrifuge tube, add 250μL of virus solution to each tube, and add 750μL TRIzol reagent, let stand at room temperature for 10 minutes; then add 200μL chloroform, vortex and oscillate for 15s, and let stand at room temperature for 10 minutes; centrifuge at 4℃12000r·min-1 for 15 minutes. After centrifugation, the solution is divided into three layers, the upper layer is a colorless aqueous phase containing RNA, and the top colorless liquid is transferred to a new 1.5mL centrifuge tube; add an equal amount of isopropanol to the colorless aqueous phase, mix well, and let stand at room temperature for 10 minutes; centrifuge at 4℃12000r·min-1 for 15 minutes, discard the supernatant, add 1mL of frozen 75% ethanol, and vortex briefly; centrifuge at 4℃, 12000r·min-1 for 5 minutes, discard the supernatant, dry in a biological safety cabinet for 5 minutes, and add 20μL of RNase-free DEPC water to fully dissolve.

[0025] Preferably, the specific method for reverse transcribing the RNA into cDNA in S12 is as follows: remove the genomic DNA, the reaction system is 1, take the PCR tube after high-pressure sterilization, prepare the system on ice, mix well and place it in a PCR instrument, and incubate at 42°C for 2 minutes; reverse transcription into cDNA: refer to reaction system 2, first react at 37°C in a PCR amplification instrument for 15 minutes, then heat to 85°C and continue to react for 5 seconds, and store the cDNA at -20°C.

[0026] Preferably, the specific method for amplifying the target gene 13 is to prepare primers: take out the primers in dry powder state, centrifuge at 4°C 4000r·min-1 for 10min, add DEPC water to dilute the primer concentration to 10μmol·L-1 for standby use, and store the rest at -20°C; amplify the target gene according to the reaction procedures of reaction systems 4 and 5;

[0027] Take 5 μL of amplified product and add 1 μL of 6× loading buffer, mix well, and perform 0.7% agarose gel electrophoresis with a sample volume of 5 μL. The electrophoresis conditions are: 100 V, 30 min;

[0028] PCR product gel recovery

[0029] Confirm the position of the target fragment under UV light, cut the fragment and cut it into pieces into a 5mL centrifuge tube, and weigh it;

[0030] Balance: Place the adsorption column in a collection tube, add 500 μL of balance solution BL, centrifuge at room temperature, 12000 r·min-1 for 1 min, and discard the waste liquid in the collection tube;

[0031] Dissolution: Add an equal volume of sol solution PN to the fragments, gently turn the centrifuge tube upside down, and dissolve at room temperature for 15 minutes;

[0032] Combination: Transfer the solution obtained in the previous step to the adsorption column in batches, let it stand at room temperature for 2 minutes, centrifuge it at 12000r·min-1 for 1 minute, and discard the waste liquid;

[0033] Cleaning: Add 600 μL of rinse solution PW to clean the adsorption column, let it stand at room temperature for 2 min, repeat the cleaning once, centrifuge at 12000 r·min-1 for 1 min, discard the waste liquid, let it stand at room temperature for 5 min to dry thoroughly;

[0034] Elution: Place the adsorption column in a new 1.5 mL centrifuge tube, drip 20 μL of ddH20 preheated at 65°C into the center of the film, let stand at room temperature for 2 min, centrifuge at 12000 r·min-1 for 2 min, and measure the concentration of the recovered gel product.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention provides an efficient method for extracting and purifying σNS protein from Nelson Bay Orthoreovirus (NBV). By constructing the pET-28a-S3 recombinant plasmid and achieving efficient expression and purification in Escherichia coli, a high-purity σNS recombinant protein with a His tag is obtained as an ideal antigen for preparing polyclonal antibodies. The optimized antibody preparation process: using the purified σNS recombinant protein as an antigen, polyclonal antibodies are prepared by immunizing rabbits. The process is simple, controllable, and has a high antibody yield. This method effectively solves the problems of low antigen purity and poor antibody specificity in the traditional antibody preparation process.

[0037] At the same time, the prepared polyclonal antibody has a high titer and shows good antibody activity. At the same time, the identification results show that the antibody has a high specific binding ability to the σNS protein and has low cross-reaction with other related proteins, ensuring the quality and availability of the antibody.

[0038] At the same time, the present invention has a wide range of application prospects. The method for preparing polyclonal antibodies to σNS of the present invention not only provides a powerful tool for in-depth research on NBV, but may also play an important role in the development of diagnostic reagents and vaccines for NBV. In addition, the method also has potential versatility and can be applied to the preparation of polyclonal antibodies to other viruses or proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the electrophoresis diagram of the target protein expression at the IPTG concentration in the embodiment of the present invention;

[0040] Figure 2The electrophoresis diagram of the supernatant, effluent, washing solution and eluate in the embodiment of the present invention;

[0041] Figure 3 The electrophoresis diagram of the supernatant, effluent, washing solution and eluate in the embodiment of the present invention;

[0042] Figure 4 This is a diagram showing the observation results under the identification imaging device in the embodiment of the present invention;

[0043] Figure 5 This is a curve diagram of absorbance value of antibody detection in an embodiment of the present invention;

[0044] Figure 6 This is a diagram showing the results of quantitative analysis of antibody specificity in an embodiment of the present invention. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Embodiment 1:

[0047] (I) Construction of pET-28a-S3 plasmid

[0048] 1. Extraction of viral RNA

[0049] (1) Take out the Nelson Bay Orthoreovirus (NBV) virus frozen at -80°C, thaw it at room temperature, place it in a 1.5 mL centrifuge tube, add 250 μL of virus solution to each tube, add 750 μL of TRIzol reagent, and let it stand at room temperature for 10 min.

[0050] (2) Add 200 μL of chloroform, vortex for 15 seconds, and let stand at room temperature for 10 minutes.

[0051] (3) Centrifuge at 4°C and 12,000 r / min for 15 min. The solution is separated into three layers. The upper layer is a colorless aqueous phase containing RNA. The top colorless liquid is transferred to a new 1.5 mL centrifuge tube.

[0052] (4) Add an equal amount of isopropanol to the colorless aqueous phase, mix well, and let stand at room temperature for 10 min.

[0053] (5) Centrifuge at 12000 r / min at 4°C for 15 min, discard the supernatant, add 1 mL of frozen 75% ethanol, and vortex briefly.

[0054] (6) Centrifuge at 4°C, 12,000 r / min-1 for 5 min, discard the supernatant, dry in a biosafety cabinet for 5 min, and add 20 μL of RNase-free DEPC water to fully dissolve. Determine the concentration of total RNA.

[0055] 2. RNA is reverse transcribed to synthesize cDNA

[0056] (1) Removal of genomic DNA: Reaction system 1 is shown in Table 1. Take a high-pressure sterilized PCR tube, prepare the system on ice, mix well, place in a PCR instrument, and incubate at 42°C for 2 min.

[0057] Table 1 Genomic gDNA reaction system

[0058]

[0059] (2) Reverse transcription to cDNA: Prepare reaction system 2 on ice according to Table 2. First, perform PCR amplification at 37°C.

[0060] The reaction was carried out for 15 min, then the temperature was raised to 85°C and the reaction was continued for 5 s, and the cDNA was stored at -20°C.

[0061] Table 2 Reverse transcription reaction system

[0062]

[0063] 3. Design and synthesize primers for target genes

[0064] According to the NBV-S3 sequence provided by the US GenBank gene library, the primers were designed using Premier 6.0 software, and appropriate restriction sites were selected according to the cloning site of the vector pET-28a. The primer design of the recombinant expression vector is shown in Table 3 and was synthesized by Shanghai Jie Li Biotechnology Co., Ltd.

[0065] Table 3 Primer sequences of recombinant expression vector

[0066]

[0067] 4. Amplify target gene

[0068] (1) Prepare primers: Take out the dry powder primers, centrifuge at 4000 r / min-1 at 4℃ for 10 min, add DEPC water to dilute the primer concentration to 10 μmol / L-1 for later use, and store the rest at -20℃.

[0069] (2) Amplify the target gene according to the reaction system in Table 4 and the reaction procedure in Table 5.

[0070] Table 4 PCR reaction system

[0071]

[0072] Table 5 PCR reaction program

[0073]

[0074]

[0075] (3) Take 5 μL of amplified product and add 1 μL of 6× loading buffer, mix well, and perform 0.7% agarose gel electrophoresis with a sample volume of 5 μL and electrophoresis conditions of 100 V for 30 min.

[0076] 5. PCR product gel recovery

[0077] (1) Confirm the position of the target fragment under ultraviolet light, cut the fragment into pieces and put them into a 5 mL centrifuge tube, and weigh them.

[0078] (2) Balance: Place the adsorption column in a collection tube, add 500 μL of balance solution BL, centrifuge at room temperature and 12000 r·min-1 for 1 min, and discard the waste liquid in the collection tube.

[0079] (3) Dissolution: Add an equal volume of sol solution PN to the fragments, and gently turn the centrifuge tube upside down.

[0080] Turn it upside down and dissolve it at room temperature for 15 minutes.

[0081] (4) Combination: The solution obtained in the previous step was transferred to the adsorption column in batches, allowed to stand at room temperature for 2 min, centrifuged at 12000 r·min-1 for 1 min, and the waste liquid was discarded.

[0082] (5) Cleaning: Add 600 μL of rinse solution PW to clean the adsorption column, let it stand at room temperature for 2 min, and repeat the cleaning once. Centrifuge at 12000 r·min-1 for 1 min, discard the waste liquid, and let it stand at room temperature for 5 min to dry thoroughly.

[0083] (5) Elution: Place the adsorption column in a new 1.5 mL centrifuge tube, drip 20 μL of ddH20 preheated at 65°C into the center of the film, let stand at room temperature for 2 min, centrifuge at 12000 r·min-1 for 2 min, and measure the concentration of the recovered gel product.

[0084] 6. Double restriction enzyme digestion of target fragment and expression vector

[0085] (1) Prepare the enzyme digestion system according to Table 6, and digest the target fragment S3 and the expression vector pET-28a with double enzymes.

[0086] Cutting treatment, reaction conditions: 37℃ 3h.

[0087] Table 6 Double enzyme digestion system

[0088]

[0089] 7. Purification of double enzyme digestion products

[0090] (1) Equilibration: Add 500 μL of equilibration solution BL to the purification column, centrifuge at room temperature and 12000 r / min-1 for 1 min, and discard the filtrate in the collection tube.

[0091] (2) Binding: Add 5 times the volume of binding solution PB and mix thoroughly. Add the resulting solution to the purification column in batches, let stand at room temperature for 2 minutes, centrifuge at 12000 r·min-1 for 1 minute, and discard the filtrate.

[0092] (3) Cleaning: Add 600 μL of rinse solution PW to the purification column, let it stand at room temperature for 2 min, centrifuge at 12000 r·min-1 for 1 min, discard the filtrate, and repeat this step once. Centrifuge at 12000 r·min-1 for 1 min, discard the waste liquid, let it stand at room temperature for 5 min to dry thoroughly.

[0093] (5) Elution: Take a 1.5 mL centrifuge tube as a new collection tube, add 20 μL of 65 °C preheated ddH20 to the middle of the adsorption membrane, let it stand at room temperature for 2 min, centrifuge at 12000 r·min-1 for 2 min, and measure the concentration of the purified product.

[0094] 8. Connect the target fragment to the expression vector

[0095] Prepare the ligation system according to Table 7 for the purified product obtained in the previous step. First, add the target gene and expression vector, incubate at 60℃ for 3 minutes, then quickly put on ice and let stand for 3 minutes. Then add Solution I and

[0096] Solution III, react at 16℃ for 30min, then react at 4℃ overnight.

[0097] Table 7 Target fragment and vector connection system

[0098]

[0099]

[0100] 9. Recombinant vector transformation

[0101] Pipette 10 μL of the ligation product and gently inject it into 50 μL of BL21 competent cells. Place on ice for 25 minutes. Heat shock in a 42°C water bath for 45 seconds, then transfer to ice and cool for 2 minutes. Add 700 μL of antibiotic-free LB liquid culture medium and incubate on a shaker for 1 hour. Centrifuge at 5000 r·min-1 for 1 minute, retain part of the supernatant to resuspend the cells, evenly spread on LB solid culture medium containing kanamycin, and incubate inverted at 37°C for 12 hours to 16 hours.

[0102] 10. Screening of positive clones

[0103] (1) Take the plate obtained in the previous step, pick 5-8 clonal colonies, add them into 10 mL of LB liquid culture medium containing kanamycin, and screen and culture them.

[0104] (2) After 12 h of incubation, 1 μL of bacterial solution was taken as a template for bacterial solution PCR. The system was the same as that in Tables 4 and 5.

[0105] (3) Plasmids were extracted from the bacterial solution that was positive for nucleic acid electrophoresis and preserved in 100% glycerol.

[0106] Label the samples and store them in a -80℃ freezer.

[0107] 11. Extraction of recombinant plasmid in small quantities

[0108] (1) Centrifuge all the positive bacterial cultures screened out at 12,000 r / min for 11 min and discard the supernatant.

[0109] (2) Add 250 μL of Buffer S1 to the precipitate and resuspend the cells without any residual clumps.

[0110] (3) Add 250 μL of Buffer S2 and immediately mix by gently inverting the tube 6-8 times. Let stand at room temperature for 3 min to disrupt the bacteria.

[0111] (4) Then add 350 μL Buffer S3, gently invert up and down 8-10 times to mix, and centrifuge at 12000 r·min-1 for 10 min.

[0112] (5) Collect the supernatant after centrifugation and place it in a new 2 mL centrifuge tube. Centrifuge at 12000 r·min-1 for 2 min, and transfer the supernatant to the adsorption column.

[0113] (6) Add 500 μL Buffer W1, let stand for 2 min, and centrifuge to discard the waste liquid.

[0114] (7) Add 500 μL Buffer W2, let stand for 2 min, centrifuge and discard the waste liquid, and repeat the wash once.

[0115] (8) 12000 r·min-1, idling for 2 min.

[0116] (9) Place the adsorption column in a new centrifuge tube, add 60 μL of ddH20 preheated at 65°C toward the middle of the adsorption membrane, let stand at room temperature for 2 min, centrifuge at 12000 r·min-1 for 2 min, and measure the concentration of the plasmid.

[0117] 12. Double enzyme digestion verification and sequencing of recombinant plasmid

[0118] The operation steps were the same as above. Double enzyme digestion was used to verify whether the S3 gene was correctly inserted into the plasmid. 40 μL of the plasmid was sent to Shanghai Jie Li Biotechnology Co., Ltd. for sequencing, and the rest was stored at -20°C.

[0119] (II) Expression and purification of His-tagged recombinant proteins

[0120] 1. Small amount of induced expression

[0121] The expression bacterial solution cultured for more than 12 hours was inoculated into the LB medium containing Kana resistance, and cultured until the OD 600 value of the bacterial solution reached 0.6-0.8. Experimental groups with different temperature gradients (16℃, 25℃ and 37℃) and different IPTG induction concentrations (0, 0.1, 0.3, 0.5, 0.7, 0.9 and 1.1mmol·L-1) were set, and cultured for 12 hours at 37℃ and 200r·min-1 on a shaker. Centrifuge at 4℃ and 8000r·min-1 for 20 minutes, discard the supernatant and collect the bacteria, and resuspend them completely in 500μL 1×PBS. Ultrasonic disruption of the bacteria in an ice water bath for about 1 minute until the bacterial solution is clear, centrifuge at 4℃ and 12000r·min-1 for 20 minutes, take the supernatant and precipitate respectively, add 4×Loading buffer, boil in water bath for 10 minutes, perform 10% SDS-PAGE electrophoresis, and detect with Coomassie Brilliant Blue staining. Figure 1 Note: M: marker, 1-7: 0, 0.1, 0.3, 0.5, 0.7, 0.9, 1.1 mM IPTG. Figure 2 Note: M: marker, 1: supernatant, 2: flow-through, 3-5: washing solution, 6-8: elution solution.

[0122] 2. Induce expression in large quantities

[0123] Select 25℃, IPTG 0.3mmol·L-1 for 12h. Collect the bacterial precipitate the next day, resuspend the bacteria with 30mL pre-cooled PBS, vortex and oscillate to mix thoroughly, add PMSF and lysozyme in turn, and let stand on ice for 30min. Ultrasonicate the bacteria, centrifuge at low temperature, and collect the supernatant.

[0124] 3. Purification

[0125] First, open the upper and lower end stoppers and use gravity to drain the protective liquid. Add 5 column volumes of Lysis Buffer to the column tube for balance. Add the supernatant to the treated gravity column, let it stand for 20 minutes, control the flow rate to slowly flow out, and collect the effluent. Add 10-15 times the column volume of Wash Buffer for washing to remove non-specifically adsorbed impurities and collect the wash solution. Add 5-10 times the column volume of Elution Buffer to elute the target protein and collect the eluate in segments. Use 3 times the column volume of Lysis Buffer and 5 times the column volume of deionized water to balance the filler in turn, store the gravity column in an equal volume of 20% ethanol and store at 4°C. Prepare samples of the supernatant, effluent, wash solution and eluate, and perform 10% SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Figure 3 ; Note: M: marker, 1: supernatant, 2: flow-through, 3-5: washing solution, 6-8: elution solution.

[0126] 4. Dialysis

[0127] Choose a dialysis bag with a suitable diameter according to the size of the target protein, cut it to about 15 cm, soak it in a beaker filled with ddH2O, and boil it for 15 minutes; take out the dialysis bag, fix one end with a clamp, pour the protein solution in and clamp the other end to ensure complete sealing. Put the dialysis bag into 1L pre-cooled PBS, place it on a magnetic stirrer and rotate it slowly at 300r·min-1, 4℃, replace PBS every 2 hours, and dialyze for 4h-8h.

[0128] 5. Concentration

[0129] Wrap the dialysis bag with PEG12000, concentrate in a 4℃ refrigerator, and observe every 30 minutes. When the volume in the dialysis bag is reduced by half, collect the concentrated protein solution, centrifuge at 4℃12000r·min-1 for 10 minutes, and take the supernatant. Detect the protein concentration, and store the rest at -80℃ after aliquoting.

[0130] 6. Detection concentration

[0131] (1) Prepare protein standard solution with a concentration of 25 mg mL-1 and dilute with PBS to a final concentration of 0.5 mg mL-1.

[0132] Prepare a 96-well plate, add 0, 1, 2, 4, 8, 12, 16, and 20 μL of standard solution in sequence, make up the volume of each well to 20 μL with PBS, and make two sets of duplicate wells.

[0133] (2) Prepare the protein working solution in a dark place.

[0134] (3) Concentration measurement: Add working solution and protein solution separately, vortex to mix, and incubate at 37°C in the dark for 30 min.

[0135] use

[0136] The A562 value was detected by ELISA instrument and the protein standard curve was drawn.

[0137] 7. Identification

[0138] (1) Load 1 μg of protein and transfer to the membrane after electrophoresis. Cut the separation gel, filter paper and PVDF membrane (methanol activated) and soak them in transfer solution. Use a wet transfer instrument to transfer the membrane, and set the parameters to 80V for 40min.

[0139] (2) Prepare 5% skim milk powder as blocking solution and block at room temperature for 2 hours on a horizontal shaker. Primary antibody overnight: dilute mouse anti-His (1:3000) with blocking solution and incubate on a shaker at 4°C; the next day, discard the primary antibody and wash the membrane 5 times with pre-cooled PBST, 10 minutes each time; secondary antibody incubation: dilute HRP-labeled goat anti-mouse IgG (1:3000) with PBST, incubate at room temperature for 1 hour, wash the membrane 5 times with PBST, 10 minutes each time; prepare the developer in a dark place and observe the results under an imager, such as Figure 4 .

[0140] (III) Preparation of polyclonal antibodies

[0141] 1. First immunization: Immunize rabbits with σNS-His recombinant protein and mix with Freund's complete adjuvant in a ratio of 1:1, totaling 400μg protein; push the syringe to slowly inject the protein solution into Freund's complete adjuvant, and push the syringe repeatedly to completely emulsify the mixture into an oil drop shape that will not diffuse in physiological saline. Wipe the rabbit skin with alcohol before injection, and select multiple subcutaneous injections on the back.

[0142] 2. Booster immunization: Booster immunization is performed every two weeks. Freund's incomplete adjuvant and protein solution are mixed in a ratio of 1:1, totaling 400 μg protein, and emulsified.

[0143] 3. Serum collection: One week after the fourth immunization, blood samples were collected from the rabbit ear vein to test serum titer and blood was collected from the heart. The blood samples were placed at 37℃ for 2 hours, centrifuged at 4℃ and 3000r·min-1 for 10 minutes, and the collected serum was placed at -80℃.

[0144] 4. Detect antibody titer

[0145] (1) Coating antigen: dilute the σNS-His recombinant protein to a concentration of 1 μg mL-1 with coating solution, and add

[0146] Pour the resulting mixture into a 96-well plate, cover with plastic wrap to prevent evaporation, and store at 4°C overnight.

[0147] (2) Washing: The next day, shake the plate to remove any residual liquid in the wells, add 200 μL of washing solution to each well, let stand for 2 min, and repeat washing 3 times. Add 200 μL of 1× BSA diluent / blocking solution and block at 37°C for 45 min.

[0148] (3) Blocking: dilute 10×BSA blocking solution to 1×BSA, 200 μL per well, incubate at 37°C for 45 min.

[0149] (4) Wash the plate and incubate with primary antibody: Take out the resistant serum obtained by immunization and dilute the serum with 1× BSA gradient.

[0150] (1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000). Add 100 μL of diluted serum to each well, set up two replicate wells, and set up a negative control group at the same time, and incubate at room temperature for 1 hour.

[0151] (5) Wash the plate and incubate with secondary antibody: dilute HRP-labeled goat anti-rabbit IgG with 1× BSA (1:10,000) and incubate at room temperature for 1 h.

[0152] (6) Color development: Add 100 μL of color development solution to each well and incubate at room temperature in the dark for 15 min.

[0153] (7) Reaction termination: Add 50 μL of stop solution to each well to stop color development, detect OD 450, record the absorbance value and draw a curve. Figure 5 .

[0154] 5. Identify antibody sensitivity and specificity

[0155] (1) S3 eukaryotic plasmid was transfected into 293T cells. After 48 h of culture, cell lysis buffer was added and the cells were collected into a centrifuge tube. After centrifugation, the supernatant was collected.

[0156] (2) The sample loading amount was 4 μg. The primary antibody was the anti-serum obtained from immunization: 5% skim milk powder was diluted in a gradient (1:1000, 1:2000, 1:4000, 1:6000, 1:8000), and mouse anti-HA (1:3000) was used as a positive control. The secondary antibodies were 1:3000 HRP goat anti-rabbit and 1:3000 HRP goat anti-mouse. Analysis Figure 6 shown.

[0157] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0158] S3 splicing sequence:

[0159]

Claims

1. A method for preparing a σNS polyclonal antibody, characterized in that: The following steps are involved: Construction of S1 and pET-28a-S3 plasmids S11. Viral RNA extraction: RNA was extracted from Nelson Bay Orthoreovirus (NBV) and purified by TRIzol lysis, chloroform extraction, and isopropanol precipitation. S12, reverse transcription of RNA into cDNA: reverse transcription of RNA into cDNA using reverse transcriptase, first remove genomic DNA, then perform reverse transcription reaction; S13, target gene amplification: primers were designed according to the NBV-S3 sequence in GenBank, and PCR amplification was performed using cDNA as a template to obtain the target gene S3 fragment. The gene S3 nucleic acid sequence is shown in SEQ ID NO: 1; S14, plasmid construction: The target gene S3 and the expression vector pET-28a were double-digested, the digested products were purified and then connected to obtain the recombinant plasmid pET-28a-S3, the recombinant plasmid was transformed into BL21 competent cells, positive clones were screened, and plasmid extraction and sequencing were performed for verification; S2. Expression and purification of His-tagged recombinant proteins S21. Small-scale induced expression: The E. coli transformed with the recombinant plasmid was induced to express at different temperatures and IPTG concentrations, and the expression products were analyzed by SDS-PAGE electrophoresis; S22, large-scale induction expression: select the optimal expression conditions for large-scale induction, collect the bacteria and lyse them to obtain a lysate containing the target protein; S23, purification: Purify the target protein using a gravity column, and obtain a highly pure His-tagged σNS recombinant protein through equilibration, loading, washing and elution steps; S24, dialysis and concentration: The purified protein is dialyzed to remove small molecule impurities and concentrated by PEG12000 to obtain a high concentration of the target protein; S3. Preparation and identification of polyclonal antibodies S31. Immunization of animals: Use purified σNS recombinant protein as antigen to immunize rabbits for the first time, and perform booster immunization regularly; S32. Collect serum: One week after the fourth immunization, collect rabbit serum, centrifuge to remove impurities and store at -80°C; S33. Antibody titer detection: The titer of antibodies in serum was detected by ELISA method to determine the activity of antibodies; S34. Identification of antibody specificity.

2. The method for preparing a σNS polyclonal antibody according to claim 1, characterized in that: The specific method for extracting viral RNA from S11 is to take out the Nelson Bay Orthoreovirus (NBV) virus frozen at -80°C, thaw it at room temperature, place it in a 1.5 mL centrifuge tube, add 250 μL of virus solution to each tube, and add 750 μL TRIzol reagent, let stand at room temperature for 10 minutes; then add 200μL chloroform, vortex and oscillate for 15s, and let stand at room temperature for 10 minutes; centrifuge at 4℃12000r·min-1 for 15 minutes. After centrifugation, the solution is divided into three layers, the upper layer is a colorless aqueous phase containing RNA, and the top colorless liquid is transferred to a new 1.5mL centrifuge tube; add an equal amount of isopropanol to the colorless aqueous phase, mix well, and let stand at room temperature for 10 minutes; centrifuge at 4℃12000r·min-1 for 15 minutes, discard the supernatant, add 1mL of frozen 75% ethanol, and vortex briefly; centrifuge at 4℃, 12000r·min-1 for 5 minutes, discard the supernatant, dry in a biological safety cabinet for 5 minutes, and add 20μL of RNase-free DEPC water to fully dissolve.

3. The method for preparing a σNS polyclonal antibody according to claim 1, characterized in that: The specific method for reverse transcribing the RNA into cDNA in S12 is as follows: remove the genomic DNA, and the reaction system is 1. Take the PCR tube after high-pressure sterilization, prepare the system on ice, mix well and place it in a PCR instrument, and incubate at 42°C for 2 minutes; reverse transcription into cDNA: refer to reaction system 2, first react at 37°C in a PCR amplifier for 15 minutes, then heat to 85°C and continue to react for 5 seconds, and store the cDNA at -20°C.

4. The method for preparing a σNS polyclonal antibody according to claim 1, characterized in that: The specific method for amplifying the target gene 13 is to prepare primers: take out the primers in dry powder state, centrifuge them at 4°C 4000r·min-1 for 10min, add DEPC water to dilute the primer concentration to 10μmol·L-1 for standby use, and store the rest at -20°C; amplify the target gene according to the reaction procedures of reaction systems 4 and 5; Take 5 μL of amplified product and add 1 μL of 6× loading buffer, mix well, and perform 0.7% agarose gel electrophoresis with a sample volume of 5 μL. The electrophoresis conditions are: 100 V, 30 min; PCR product gel recovery Confirm the position of the target fragment under UV light, cut the fragment and cut it into pieces into a 5mL centrifuge tube, and weigh it; Balance: Place the adsorption column in a collection tube, add 500 μL of balance solution BL, centrifuge at room temperature, 12000 r·min-1 for 1 min, and discard the waste liquid in the collection tube; Dissolution: Add an equal volume of sol solution PN to the fragments, gently turn the centrifuge tube upside down, and dissolve at room temperature for 15 minutes; Combination: Transfer the solution obtained in the previous step to the adsorption column in batches, let it stand at room temperature for 2 minutes, centrifuge it at 12000r·min-1 for 1 minute, and discard the waste liquid; Cleaning: Add 600 μL of rinse solution PW to clean the adsorption column, let it stand at room temperature for 2 min, repeat the cleaning once, centrifuge at 12000 r·min-1 for 1 min, discard the waste liquid, let it stand at room temperature for 5 min to dry thoroughly; Elution: Place the adsorption column in a new 1.5 mL centrifuge tube, drip 20 μL of ddH20 preheated at 65°C into the center of the film, let stand at room temperature for 2 min, centrifuge at 12000 r·min-1 for 2 min, and measure the concentration of the recovered gel product.

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