CN108484736A - Expression of tembusu virus non-structural protein NS2A truncated proteins and products thereof and application - Google Patents

Abstract

Then the present invention relates to a kind of expressions of tembusu virus non-structural protein NS2A truncated proteins and products thereof and application to be recombinantly expressed by truncating non-structural protein NS2A, expression is easy after truncation, and expression quantity is high；Recombinant protein is set to be expressed in inclusion body by Optimal Expression condition in expression, convenient for purifying, the albumen of purifying has immunogenicity, the high polyvalent antibody of potency can be obtained after immune animal, and antibody high sensitivity obtained, can be used for tembusu virus non-structural protein NS2A detection and and detect tembusu virus in infection different times non-structural protein NS2A positioning situations of change in the cell, be of great significance to the research of tembusu virus.

Description

Method for expressing Tembusu virus non-structural protein NS2A truncated protein and its products and application

technical field

The invention belongs to the field of biotechnology, and relates to a method for expressing a non-structural protein NS2A truncated protein of Tambusu virus, and also relates to the expressed truncated protein and its application in preparing polyclonal antibodies.

Background technique

Tembusu virus disease (Tembusu virus disease) is an acute infectious disease caused by Tembusu virus (Tembusu virus, TMUV). Ovarian inflammation and neurological symptoms, the main clinical symptoms are loss of appetite or abolition, a sharp drop in body weight and a drop in egg production. With the continuous expansion of poultry industry in my country, Tambusu virus, as one of the main viruses threatening poultry industry in my country, has caused huge economic losses. However, there is no specific drug and treatment plan suitable for the treatment of Tembusu virus infection.

Tembusu virus non-structural protein NS2A plays an important role in virus gene replication and virus assembly, but so far, the function and localization of non-structural protein NS2A have not been studied in detail, and the use of tagged antibodies does not matter It is very limited in the study of localization or its function, and the full-length NS2A protein is a transmembrane protein with eight transmembrane regions and contains many rare codons, so we provide a truncation scheme to facilitate protein identification Expression, and the antibody prepared at the same time can recognize the full-length NS2A protein with high specificity and high sensitivity. The polyclonal antibody of NS2A plays an important role in detecting the localization of NS2A during DTMUV infection.

Therefore, there is an urgent need for an easily expressed and immunogenic NS2A truncated fragment, which is of great significance for the prevention and treatment of Tembusu virus.

Contents of the invention

In view of this, one of the purposes of the present invention is to provide a method for expressing the non-structural protein NS2A truncated protein of Tambusu virus; the second purpose of the present invention is to provide the non-structural protein of Tambusu virus obtained by the expression method. Structural protein NS2A truncated protein; the third object of the present invention is to provide the application of the tampusu virus non-structural protein NS2A truncated protein in the preparation of anti-Tambusu virus non-structural protein NS2A polyclonal antibody; the purpose of the present invention The fourth is to provide the anti-Tambusu virus non-structural protein NS2A polyclonal antibody made by using the tampusu virus non-structural protein NS2A truncated protein; the fifth object of the present invention is to provide the anti-Tembusu virus non-structural protein NS2A polyclonal antibody Application of polyclonal antibody against structural protein NS2A in detection of Tembusu virus infection.

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

1. The method for expressing the truncated protein of the nonstructural protein NS2A of Tambusu virus, the specific steps are as follows:

1) Use the RNA extracted from duck embryo fibroblasts infected with Tambusu virus as a template, reverse transcribe and synthesize cDNA, then use the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers to perform PCR amplification, and amplify Increase the NS2A truncated gene;

2) Connect the NS2A truncated gene amplified in step 1) into an expression vector to obtain a recombinant expression vector, then transform the recombinant expression vector into a host expression bacterium to obtain an engineered bacterium, then induce expression of the engineered bacterium with IPTG, and obtain the recombinant expression vector after purification Bushu virus nonstructural protein NS2A truncated protein.

Preferably, in step 2), the expression vector is pGEX-4T-1 plasmid.

Preferably, in step 2), the host expression bacteria is BL21(DE3).

Preferably, in step 2), the induced expression is to add IPTG at a final concentration of 0.4-0.5 mM when the engineered bacteria are cultivated to A600=0.6, and induce the expression for 6-8 hours.

Preferably, the purification is to take the induced expression bacterial liquid, collect the bacterial cells by centrifugation, and then add a Tris-HCl resuspended bacterial body with a concentration equivalent to 1/10 of the bacterial liquid volume as 20mM and a pH of 8, and take the resuspended bacterial liquid , add 6×loading buffer equivalent to 1/5 of the volume of the resuspended bacteria, heat and denature in a water bath at 100°C for 10 minutes, and then use 12% SDS-PAGE gel electrophoresis. After electrophoresis, use 0.25mMol/L KCL solution for color development. Cut the gel to recover the target fragment, and use the dialysis bag to obtain the NS2A truncated protein of Tambusu virus.

2. The Tembusu virus non-structural protein NS2A truncated protein obtained by the expression method.

3. Application of the truncated protein of Tembusu virus non-structural protein NS2A in the preparation of anti-Tembusu virus non-structural protein NS2A polyclonal antibody.

4. An anti-Tambusu virus non-structural protein NS2A polyclonal antibody prepared by using the Tembusu virus non-structural protein NS2A truncated protein.

5. Application of the anti-Tembusu virus non-structural protein NS2A polyclonal antibody in detecting Tambusu virus infection.

The beneficial effect of the present invention is that: the present invention discloses the expression method of the non-structural protein NS2A truncated protein of Tambusu virus, by truncating the non-structural protein NS2A, it is easy to express, and the expression amount is high; by optimizing the expression conditions, the The recombinant protein is expressed in the inclusion body, which is easy to purify. The purified protein is immunogenic. After immunizing animals, high-titer polyantiserum can be obtained, and the prepared antibody has high sensitivity. It can be used for the non-structural protein NS2A of Tembusu virus The detection of Tembusu virus, and the change of localization of non-structural protein NS2A in cells during different infection periods of Tembusu virus, so the research on Tembusu virus is of great significance.

Description of drawings

In order to make the purpose, technical scheme and beneficial effect of the present invention clearer, the present invention provides the following drawings for illustration:

Figure 1 is the verification and time optimization of NS2A truncated protein expression (A: truncated expression map; M is Marker, 1 is no-load induction for 6 h, 2 is no-load induction for 8 h, 3 is no-load induction for 10 h, and 4 is no-load induction. Induced for 6 hours, 5 for 8 hours without induction, 6 for 10 hours without induction, 7 for 6 hours induced, 8 for 8 hours induced, 9 for 10 hours induced; B is the full-length expression map: M is Marker, 1 is empty induction for 6 hours, and 2 is No-load induction for 8h, 3 for 6h without induction, 4 for 8h without induction, 5 for 10h without induction, 6 for 6h induction, 7 for 8h induction, and 8 for 10h induction).

Figure 2 shows the samples after induced expression were verified by western blot (M is a marker, negative is a blank control, and the whole bacteria is a sample 6 hours after induction).

Figure 3 is the position for verifying the expression of the target protein (negative is empty load, whole bacteria is whole bacteria after expression, supernatant is the supernatant after ultrasonic centrifugation, and precipitate is the precipitate after ultrasonic centrifugation including inclusion bodies).

Figure 4 is the verification of the purified protein (A: SDS-PAGE image of the purified protein to verify the purity of the purified protein, B: WB image of the purified protein to verify whether the purified protein is the target protein).

Figure 5 is a graph showing the results of the antiserum titer test, when the ratio of positive to negative is greater than 2, it is positive.

Figure 6 is the use of the prepared polyclonal antibody to explore the intracellular localization of NS2A during DTMUV infection (the localization of NS2A protein at the NS2A position, DAPI is the localization of the nucleus, and merge is the localization of the two pictures after merging).

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Embodiment 1, the preparation of cDNA template

1. Proliferation culture of TMUV

The TMUV-CQW1 seed virus was inoculated into the dense monolayer duck embryo fibroblasts (DEF) that had just grown, discarded the virus solution after adsorption at 37°C for 1 hour, and then added calf serum with a volume fraction of 2% and 100IU/mL double Anti-(penicillin, streptomycin) DMEM was used to maintain the nutrient solution, and then cultured at 37°C for 24-48 hours.

2. Extraction of total cellular RNA

1) Select DEF (100mL cell bottle) with a cytopathic effect (CPE) of 70% after TMUV seed virus infection;

2) Pour off the cell culture medium, add 1ml RNAiso ^TM plus to resuspend the cells, and react at room temperature (18-25°C) for several minutes;

3) Add 100 μl BCP to each sample, mix manually for 30 seconds, place on ice for 10 minutes, and centrifuge at 12,000 g for 15 minutes at 4°C;

4) Carefully pipette the transparent supernatant into a new RNase-free EP tube, add 500 μl of isopropanol to each tube, gently invert up and down until mixed, place at room temperature for 10 minutes, and centrifuge at 4°C and 12000g for 10 minutes;

5) Remove the supernatant (be careful not to pour out the white precipitate), add 1ml of 75% ethanol (prepared with DEPC water) to the original EP tube, mix up and down, and centrifuge at 4°C, 7500g for 5min;

6) remove supernatant, repeat step 4);

7) Dry at room temperature for several minutes until the precipitate is transparent, add 20 μl DEPC water to each tube, flick to help dissolve;

8) Take 1 μl of extracted total RNA to detect its RNA concentration with a Nanodrop 2000 protein and nucleic acid concentration detector. The RNA quality is measured by the value of OD260/OD280 between 1.8 and 2.0.

3. Reverse transcription to synthesize cDNA first strand

Take the pre-cooled RNase-free PCR tube and add it to the pre-reaction system: 2 μg RNA (convert the volume according to the measured concentration), 1 μl Oligo(dT)18, add RNase-free ddH ₂ O to make up 8 μl, pipette the tip to mix, and immediately Centrifuge, incubate in PCR instrument at 65°C for 5min, take out and chill on ice for 2min. Continue to add 10 μl of 2RT Mix and 2 μl of HisCript Enzyme Mix to the original tube, so that the final volume of each tube is 20 μl, mix well, spin off, react on the PCR instrument at 25°C for 5 minutes, at 50°C for 45 minutes, and finally at 85°C The reaction was done for 5 minutes, and the synthesized cDNA was taken out and stored in a -80°C refrigerator for later use.

Embodiment 2, TMUV CQW1 truncated NS2A gene fragment amplification

1. Primer Design

According to the sequence of the TMUV CQW1 strain uploaded to GenBank (Genbank: KM233707), combined with the restriction site on the pGEX-4T-1 vector, Primer 5 was used to design a primer for the truncated NS2A gene fragment:

NS2A-F: 5'-ccgcgtggatccccg gaattc ctagtggcagctgcattt-3' (SEQ ID NO.1) (underlined italics are EcoRI restriction sites);

NS2A-R: 5'-gtcacgatgcggccg ctcgag gtggtggtggtggtggtg aaaaggagctaaccgtccc-3' (SEQ ID NO.2) (underlined italics are XhoI restriction sites);

NS2A-F and NS2A-R are respectively used as upstream and downstream primers to amplify the truncated NS2A gene fragment (60-150aa), covering the gene fragment of NS2A gene from 178bp-450bp. The primers were synthesized by Qingke Biotechnology Co., Ltd.

Simultaneously design primers for amplifying the full length of the NS2A gene. The specific primer sequences are as follows:

NS2Afull-F: 5'-ccgcgtggatccccg gaattc tttcaaggggggtggcatgg-3' (SEQ ID NO.3) (underlined italics are EcoRI restriction sites);

NS2Afull-R: 5'-gtcacgatgcggccg ctcgag gtggtggtggtggtggtgtctccgtgtcactggcttc-3' (SEQ ID NO.4) (the underlined italic is the XhoI restriction site);

2. PCR reaction

The PCR reaction system is as follows:

The PCR reaction procedure is as follows:

Step 1: 98℃, 2min;

Step 2: 98°C, 10s; 59°C, 15s; 72°C, 5s; (30 cycles)

Step 3: 72°C, 5min.

The PCR amplification results were observed by 2% agarose gel electrophoresis; the specific target bands were recovered according to the instructions of the TIANGEN DNA Purification and Recovery Kit, and the PCR amplification products were stored at -20°C for future use.

Example 3, pGEX-4T-NS2A _60-150aa recombinant prokaryotic expression plasmid construction

Double enzyme digestion pGEX-4T-1 empty plasmid, the enzyme digestion system is as follows:

After reacting at 37°C for 15 min, linear DNA fragments were recovered with TIANGEN DNA Purification and Recovery Kit.

use II One Step Cloning Kit kit, connect the purified truncated NS2A gene fragment to the pGEX-4T-1 empty plasmid after double digestion. The reaction system is as follows:

Note: Optimum amount of cloning vector used = [0.02 base pairs of cloning vector] ng (0.03pmol)

Optimum amount of inserts used = [0.04 base pairs of inserts] ng (0.06 pmol)

After reacting the reaction system at 37°C for 30 minutes, place it on ice, take 10 μl of the ligation product and transform it into 100 μl of competent cells expressing the strain BL21(DE3), pick positive clones on the Amp plate for expansion, and extract the plasmid for sequencing. The recombinant plasmid was named pGEX-4T-1-NS2A.

The full-length sequence of NS2A was ligated into pGEX-4T-1 according to the same method as above, and the obtained recombinant plasmid was named: pGEX-4T-1-NS2A'.

Example 4, Induced expression of TMUV truncated NS2A gene recombinant prokaryotic expression protein

Transform the correctly identified recombinant plasmids pGEX-4T-1-NS2A and pGEX-4T-1-NS2A' into BL21(DE3) host expression bacteria, pick a single clone and inoculate it into LB liquid medium containing 50 μg/mL Amp for overnight culture The next day, take the overnight bacterial solution and inoculate it into a new LB medium containing 50 μg/mL Amp to make the culture solution A ₆₀₀ = 0.05 after adding the bacterial solution, and continue to cultivate until A ₆₀₀ ≈ 0.6, and the final concentration is 0.4 mM and 0.5mM isopropyl-BD-thiogalactopyranosyl (IPTG) was added to induce expression, and the bacterial cells were collected by centrifugation at 6h, 8h and 10 hours of expression, respectively. Add 20 mM Tris-HCL (pH=8) to resuspend the bacteria according to one-tenth of the original LB amount. Take 100 μL of the resuspension solution and add 20 μL of 6× protein loading buffer, heat and denature in a water bath at 100°C for 10 minutes, perform 12% SDS-PAGE gel electrophoresis, stain with Coomassie brilliant blue, and observe the expression results. Results The fusion protein of about 35kDa was expressed after the recombinant plasmid was induced, and there was no corresponding band in the empty vector group and the NS2A gene full-length group (Fig. 1).

Example 5, Identification of pGEX-4T-NS2A _60-150aa recombinant prokaryotic expressed protein

Using Western-blotting analysis, the steps are as follows:

1) The expressed protein was separated by SDS-PAGE electrophoresis, transferred to PVDF membrane, and blocked with TBST containing 5% (w/v) skimmed milk powder at 37°C for 2 hours with slow shaking;

2) After washing the membrane 3 times with TBST (5 min/time), add mouse anti-His antibody diluted in TBST containing 5% (w/v) skim milk powder, and incubate at 37°C for 2 h;

3) After washing the membrane 3 times with TBST (5 min/time), add HRP-labeled goat anti-mouse IgG secondary antibody diluted in TBST containing 5% (w/v) skimmed milk powder, and incubate at 37°C for 1 h;

4) After washing the membrane 3 times with TBST (5 min/time), the ECL color development kit was used for color development, and specific reaction bands could be observed as a result (Figure 2).

Example 6, Purification of pGEX-4T-NS2A _60-150aa recombinant prokaryotic expression protein

It has been identified that the pGEX-4T-NS2A _60-150aa recombinant prokaryotic expression protein is expressed in bacterial inclusion bodies (Figure 3), so after the inclusion bodies containing the target gene protein are used for SDS-PAGE gel electrophoresis, use pre-cooled 0.25mMol /L KCl solution for color development, the gel was cut to recover the target fragment, and the purified prokaryotic expression protein of the truncated NS2A gene of Tambusu virus was obtained by using a dialysis bag. The specific operation is as follows:

1) Gel compounding: 4 mL of separating gel, flattened with absolute ethanol, and 0.8 mL of concentrated gel, flattened with absolute ethanol;

2) Electrophoresis: add 0.6-1mL sample to each gel;

3) Treatment of the dialysis bag: prepare a solution: 2% NaHCO ₃ , 1mM EDTA, set the volume to 1L, boil it and put it into the dialysis bag for 10 minutes, put it into RO water and place it at 4°C for use;

4) Gel cutting: prepare 0.25mM KCl solution, pre-cool it as a color developing solution, peel off the glue and put it into the color developing solution, a white band can be seen at the protein, cut off the glue and put it in Add Tris-glycine buffer solution (0.5 mL for one gel) to the dialysis bag to ensure no leakage;

5) Electroelution: fill the electrophoresis tank with Tris-glycine buffer, put the dialysis bag with gel blocks into the electrophoresis tank, make the direction of the dialysis bag perpendicular to the current direction, 120V for two hours, reverse the electrode and then 120V After running for 2 minutes, after electrophoresis, the liquid in the dialysis bag was sucked into the EP tube and stored at -80°C for later use.

After the purified TMUV truncated NS2A prokaryotic expression protein was analyzed by SDS-PAGE and Western Blot, the results are shown in FIG. 4 . The results showed that the target protein was successfully obtained.

Example 7, Preparation of polyclonal antibody of TMUV truncated NS2A gene

1. Preparation of Animal Experiments

Six 6- to 8-week-old Kunming mice were purchased, and one was randomly selected for eye socket blood collection. Place at room temperature (18-25°C) for 1 hour, then transfer to 4°C for overnight, centrifuge at 3000rmp for 10 minutes at 4°C, and separate the negative serum. The remaining 5 Kunming rats were used as the experimental group.

2. Determination of Purified Protein Concentration

The concentration of the purified truncated NS2A prokaryotic expression protein was determined using the Pierce ^TM BCA Protein Assay Kit kit from Thermo Scientific, and the specific operations were as follows:

1) Dilute standard BSA with elution buffer (excluding urea) to final concentrations of 2000 μg/ml, 1500 μg/ml, 1000 μg/ml, 750 μg/ml, 500 μg/ml, 250 μg/ml and 125 μg/ml Liquid, the volume of each concentration is 40 μl;

2) According to the number of samples to be measured and the sum of the number of standard products with different concentrations, prepare the working solution according to the ratio of solution A: solution B = 50:1;

3) Add 10 μl of the above-mentioned diluted standard substance and sample to be tested into EP tubes, and add 200 μl of the prepared working solution to each tube;

4) Incubate at 37°C for 30 minutes. After cooling, measure the absorbance value at a wavelength of 562nm with a Nanodrop 2000 protein and nucleic acid concentration detector. First, measure the concentration of the diluted standard to establish a standard curve. According to the standard curve, the concentration of the sample to be tested was obtained, and the concentration was 1 mg/mL.

3. Preparation of TMUV truncated NS2A gene polyantiserum by immunizing Kunming mice

1) The first immunization: according to the protein amount of 100 μg per mouse, emulsify the purified protein with Freund's complete adjuvant at a ratio of 1:1, and emulsify to the water-in-oil state, that is, drop it into water for 1 minute without dispersing That is, the emulsification is complete, and multi-point subcutaneous injection of Kunming mice (200 μL/only);

2) The second immunization: 2 weeks after the first immunization, the same amount of emulsified purified protein and incomplete adjuvant were emulsified and purified according to the protein amount of 100 μg per mouse. );

3) The third immunization: three weeks after the first immunization, the immunization procedure is the same as that of the second immunization;

4) The fourth booster immunization: 4 weeks after the first immunization, the immunization procedure was the same as that of the second immunization. On the 10th day after immunization, the eyeballs were removed and a large amount of blood was collected to separate the serum;

Example 8, Polyclonal antibody titer determination of TMUV truncated NS2A gene

The polyclonal antibody titer of the purified TMUV truncated NS2A gene was determined by indirect ELISA, and the specific steps were as follows:

1) Antigen coating: the purified protein was diluted to 10 μg/mL with sodium carbonate-sodium bicarbonate buffer as a coating solution, 100 μL was added to each well, and coated overnight at 4°C.

2) Seal the enzyme-labeled reaction wells: wash three times with PBST, 5min/time, pat dry and then use 5% BSA as a blocking solution, add 100μL to each well, block at 37°C for 40min, wash three times with PSBT after the sealing is completed, Pat dry.

3) Add the sample to be tested: after doubling the antiserum and negative serum, add 100 μL to each well, react at 37°C for 1 hour, wash with PBST three times after the reaction, and pat dry.

4) Add enzyme-labeled antibody: Dilute the HRP-labeled goat anti-mouse secondary antibody at 1:500 according to the instruction manual, add 100 μL of diluent to each well, react at 37°C for 1 hour, wash with PBST three times, and pat dry.

5) Add the substrate reaction solution: TMB-hydrogen peroxide urea solution is used as the reaction solution, add 100 μL to each well, and place it at 37°C in the dark for 5 minutes.

6) Stopping the reaction: 50 μL of stop solution was added to each well to stop the reaction.

7) Determination of absorbance: after the reaction was terminated, the absorbance was measured at a wavelength of 450 nm. The ratio of the absorbance of the sample well to the negative control was greater than 2 as the titer of the antibody. The results are shown in Figure 5. The results showed that the final polyclonal titer was 1:128000.

Example 9. Indirect immunofluorescence detection of NS2A protein localization changes during virus infection

1) Virus-infected cells: culture duck embryo fibroblasts on a 6-well plate, put cell slides in each well, and when the cells grow to a suitable density, infect with DTMUV, respectively, at 12, 24, 36, At 48, 60 and 72 hours, the slides were taken out and fixed with 4% paraformaldehyde.

2) Permeabilization: After all the samples at all time points are collected, wash with PBST three times after fixation, then permeabilize with pre-cooled 0.22% Triton X-100, permeabilize at 4°C for 20min, and then Wash three times with PBST.

3) Blocking: block with 5% BSA at 37° C. for 30 min, and wash with PBST three times after the blocking is completed.

4) Incubate the primary antibody: Dilute the prepared mouse anti-NS2A polyclonal antibody 1:500 with 1% BSA, incubate overnight at 4°C, and wash three times with PSBT.

5) Incubate with secondary antibody: Incubate with 1:1000 diluted FITC-goat anti-mouse secondary antibody at 37°C for 2 hours and then use PBST three times.

6) Nuclei staining, mounting: Dilute DAPI 1:1000, incubate for 10 min, wash three times with PBST, and seal.

7) Observation: observe with a fluorescence microscope, green fluorescence indicates the location of NS2A in the cell, blue fluorescence indicates the position of the nucleus, and take pictures, the results are shown in Figure 6. The results showed that NS2A first localized around the nucleus when DTMUV infected cells, and gradually began to diffusely distribute in the cytoplasm of the whole cell as the infection time increased.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that it can be described in terms of form and Various changes may be made in the details without departing from the scope of the invention defined by the claims.

sequence listing

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Claims (9)

1. the expression method of nonstructural protein NS2A truncated protein of Tambusu virus, it is characterized in that, concrete steps are as follows: 1) Use the RNA extracted from duck embryo fibroblasts infected with Tambusu virus as a template, reverse transcribe and synthesize cDNA, then use the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers to perform PCR amplification, and amplify Increase the NS2A truncated gene; 2) Connect the NS2A truncated gene amplified in step 1) into an expression vector to obtain a recombinant expression vector, then transform the recombinant expression vector into a host expression bacterium to obtain an engineered bacterium, then induce expression of the engineered bacterium with IPTG, and obtain the recombinant expression vector after purification Bushu virus nonstructural protein NS2A truncated protein. 2. The method for expressing the nonstructural protein NS2A truncated protein of Tambusu virus according to claim 1, characterized in that: in step 2), the expression vector is the pGEX-4T-1 plasmid. 3. The method for expressing the nonstructural protein NS2A truncated protein of Tambusu virus according to claim 1, characterized in that: in step 2), the host expression bacterium is BL21 (DE3). 4. according to the expression method of the said Tembusu virus nonstructural protein NS2A truncated protein of claim 1, it is characterized in that: in step 2), described induction expression is when engineering bacterium is cultivated to A600=0.6, by final concentration Add IPTG at 0.4-0.5mM to induce expression for 6-8 hours. 5. according to the expression method of the said Tembusu virus nonstructural protein NS2A truncated protein of claim 1, it is characterized in that: described purifying is to get induced expression bacterium liquid, centrifugal collection thalline, then add and be equivalent to bacterium liquid volume 1 Tris-HCl with a concentration of 20mM and a pH of 8/10 resuspended bacteria, took the resuspended bacteria, added 6×loadingbuffer equivalent to 1/5 of the volume of the resuspended bacteria, heated and denatured in a water bath at 100°C for 10 minutes before use 12% SDS-PAGE gel electrophoresis, after electrophoresis, use 0.25mMol/L KCL solution to develop color, cut the gel to recover the target fragment, and use the dialysis bag to obtain the NS2A truncated protein of Tambusu virus. 6. The Tembusu virus non-structural protein NS2A truncated protein obtained by the expression method described in any one of claims 1-5. 7. The application of the Tembusu virus non-structural protein NS2A truncated protein of claim 6 in the preparation of anti-Tembusu virus non-structural protein NS2A polyclonal antibody. 8. the anti-Tambusu virus non-structural protein NS2A polyclonal antibody that utilizes the Tembusu virus non-structural protein NS2A truncated protein of claim 6 to make. 9. The application of the anti-Tembusu virus non-structural protein NS2A polyclonal antibody described in claim 8 in the detection of Tembusu virus infection.