A method for preparing recombinant duck Tembusu virus E protein domain III protein

Abstract

The present invention discloses a method for preparing a recombinant duck Tembusu virus E protein domain III protein. The present invention first provides a Pichia pastoris GS115 strain for preparing duck Tembusu virus E protein domain III. The recombinant strain is obtained by intercepting the duck Tembusu virus envelope protein EDIII antigen region, optimizing the codons to obtain the coding sequence of the duck Tembusu virus EDIII protein, and then cloning it into a yeast expression vector pPIC9K and transforming it into a yeast host strain GS115. Under the action of methanol, the duck Tembusu virus EDIII protein is efficiently expressed. At the same time, the protein is used as a coating antigen for a duck Tembusu virus antibody indirect ELISA diagnostic kit, which has high specificity, repeatability and stability, and can be used for the diagnosis of related diseases caused by Tembusu virus.

Description

Technical Field

[0001] The invention belongs to the field of veterinary biotechnology, and specifically relates to yeast expression and application of duck Tembusu virus EDⅢ protein. Background Art

[0002] Duck Tembusu virus belongs to the Flaviviridae family, Flaviviridae genus, and Entayavira group. Tembusu virus was first isolated from Aedes and Culex mosquitoes in Malaysia and Thailand. Although birds and poultry were once considered the natural vertebrate hosts of the virus, there are few reports of avian animal diseases caused by the virus. It is still unknown how waterfowl such as ducks and geese are infected with the disease, and it is estimated that it is related to mosquitoes. At the same time, there are literature reports that Tembusu virus can be detected in dead sparrows in duck farms, suggesting that the virus can be transmitted by birds. There are also literature reports that the virus can be isolated from the cloaca of infected ducks, indicating that the virus can be excreted through feces, contaminating the environment, feed, drinking water, utensils, etc., thereby causing the spread of the virus.

[0003] The E protein is the main surface structural protein of duck Tembusu virus, which is composed of 500 amino acid residues and contains many antigenic epitopes related to host tropism, host cell membrane fusion and host cell surface receptor binding. According to its different functions, the E protein is divided into three domains (I, II and III), and the immunoglobulin-like third domain (EDIII) is located in the outermost layer of the virus, which plays an important role in mediating the binding of the virus to the host receptor and is also the dominant epitope region for inducing neutralizing antibodies. Therefore, the expression and function of EDIII are of great significance to the interaction between the virus and the host cell, clinical diagnosis and subunit vaccine research.

[0004] The diagnosis and prevention of new epidemic diseases are the key issues that scientific researchers need to solve first. The establishment of antibody detection methods for the virus is very meaningful for epidemiological investigations and evaluation of immune effects of the disease. ELISA is very popular due to its large detection throughput, accurate results, high sensitivity, and easy promotion at the grassroots level, and has become the most widely used serological detection method. Therefore, the present invention uses duck Tembusu virus EDIII gene as a template, collects protein through yeast expression, and then uses purified recombinant EDIII protein as an antigen to establish an indirect ELISA detection method, which provides technical means for early diagnosis and large-scale detection of the virus. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a yeast expression method for duck Tembusu virus EDIII protein, collect duck Tembusu virus EDIII protein as a coating antigen through yeast expression, and use it in a Tembusu virus antibody indirect ELISA diagnostic kit to improve the specificity, repeatability and stability of the kit.

[0006] Another object of the present invention is to provide a use of duck Tembusu virus EDIII protein expressed by yeast in an indirect ELISA diagnostic kit for duck Tembusu virus antibodies.

[0007] The present invention is achieved through the following technical solutions:

[0008] Duck Tembusu virus EDIII protein, the amino acid sequence of which is shown in SEQ ID NO.2:

[0009] Met Gln Gly Leu Lys Leu Lys Gly Met Thr Tyr Pro Met Cys Ser Asn ThrPhe Ser Leu Val Lys Asn Pro Thr Asp Thr Gly His Gly Thr Val Val Val Glu LeuSer Tyr Ala Gly Thr Asp Gly Pro Cys Arg Val Pro Ile Ser Met Ser Ala Asp LeuAsn Asp Met Thr Pro Val Gly Arg Leu Ile Thr Val Asn Pro Tyr Val Ser Thr SerSer Thr Gly Ala Lys Ile Met Val Glu Val Glu Pro Pro Phe Gly Asp Ser Phe IleLeu Val Gly Ser Gly Lys Gly Gln Ile Arg Tyr Gln Trp His Arg Ser Gly Ser ThrIle Gly Lys Ala Phe Thr Ser Thr Leu Lys Gly Ala His His His

[0010] The present invention further provides a method for preparing a recombinant duck Tembusu virus EDIII protein, comprising the following steps:

[0011] (1) Construction of expression vector: The codon-optimized duck Tembusu virus EDIII gene shown in SEQ ID NO.1 was artificially synthesized, and restriction endonuclease cleavage sites were designed on the gene sequence. The EDIII gene sequence and the yeast expression vector pPIC9K were digested with the same restriction endonuclease and then connected together. The clones containing the EDIII gene sequence were detected and screened by double enzyme digestion;

[0012] (2) Construction of yeast expression system: The recombinant expression plasmid was linearized using restriction endonucleases, and the gene sequence of EDIII was integrated into the yeast genome by electroporation to obtain the recombinant yeast strain GS115 / pPIC9K-EDIII; the transformed bacterial suspension was spread on MD plates and cultured at 30°C; single colonies were picked for screening and identification, and the obtained single colonies were induced for expression;

[0013] (3) Purification of recombinant protein.

[0014] As a preferred embodiment of the present invention, the preparation method comprises the following steps:

[0015] (1) Construction of expression vector: A restriction endonuclease cleavage site was designed on the artificially synthesized duck Tembusu virus EDIII gene sequence. The EDIII gene sequence and the yeast expression vector pPIC9K were digested with the same restriction endonuclease and then connected together. The clones containing the EDIII gene sequence were detected and screened by double enzyme digestion.

[0016] (2) Construction of yeast expression system: The recombinant expression plasmid was linearized using restriction endonucleases, and the EDIII gene sequence was integrated into the yeast genome by electroporation. The transformed bacterial suspension was spread on MD plates and cultured at 30°C. Single colonies were picked for G418 resistance screening and PCR colony identification. The resulting single colonies were induced to express at a small dose, and the expression supernatant was detected by SDS-PAGE to confirm positive clones.

[0017] (3) Purification and activity detection of recombinant proteins: Positive clones were selected for high-dose induced expression, and the expression supernatant was purified using Protein Pure Ni-NTA resin. The purified recombinant protein was detected by SDS-PAGE and Western blot.

[0018] As a preferred embodiment of the present invention, the yeast cell in step (2) is GS115.

[0019] As a preferred embodiment of the present invention, the recombinant yeast strain GS115 / pPIC9K-EDIII is a high-copy positive recombinant yeast obtained by PCR identification and G418 resistance plate screening. The universal primers for PCR identification are 5′AOX1 and 3′AOX1, and their sequences are 5′-GACTGGTTCCAATTGACAAGC-3′ and 5′-GCAAATGGCATTCTGACATCC-3′, respectively.

[0020] As a preferred embodiment of the present invention, the recombinant yeast is Mut + Phenotyping of recombinant yeast.

[0021] As a preferred embodiment of the present invention, the recombinant yeast strain is fermented and cultured by the following method: the recombinant yeast strain GS115 / pPIC9K-EDIII is cultured in BMGY medium at 30°C and 220rpm for 24 hours, centrifuged, the supernatant is discarded, 10mL BMMY liquid medium is added, and the culture is carried out at 30°C and 250rpm for 5 days, and methanol is supplemented every 24 hours to maintain the methanol concentration at 1%; or the recombinant yeast strain GS115 / pPIC9K-EDIII is cultured in BMGY medium until OD600 is 3, the bacteria are collected by centrifugation, transferred to a conical flask, BMMY medium is added to dilute to OD600 of 1, sealed with 8 layers of sterile gauze, and cultured on a shaker at 30°C and 220rpm for 96 hours, and 100% methanol is added every 24 hours to a final concentration of 1%.

[0022] As a preferred embodiment of the present invention, the recombinant duck Tembusu virus E protein domain III protein is separated and purified using a Ni-NTA Resin column.

[0023] A recombinant yeast expression plasmid for highly expressing duck Tembusu virus EDⅢ protein in yeast. The recombinant yeast expression plasmid is obtained by serially connecting the codon-optimized duck Tembusu virus EDⅢ gene shown in artificially synthesized SEQ ID NO.1 between two restriction endonuclease sites, EcoRⅠ and NotⅠ, in a yeast expression vector pPIC9K.

[0024] A recombinant yeast strain for highly expressing duck Tembusu virus EDⅢ protein in yeast. The recombinant yeast strain is obtained by taking yeast cell GS115 as host cell and transfecting the recombinant yeast expression plasmid of the invention.

[0025] The invention discloses an application of the recombinant yeast expression plasmid and the recombinant yeast strain in the high-efficiency expression and preparation of duck Tembusu virus EDⅢ protein.

[0026] The invention discloses an application of the duck Tembusu virus EDIII protein expressed by yeast as a coating antigen in an indirect ELISA diagnostic kit for Tembusu virus antibodies.

[0027] The invention discloses an indirect ELISA diagnostic kit for Tembusu virus antibody, comprising an ELISA plate coated with duck Tembusu virus EDIII protein expressed by the method of the invention as an antigen, 100 μl duck Tembusu virus positive control serum, 100 μl duck Tembusu virus negative control serum, 100 μl HRP-labeled goat anti-duck secondary antibody, 100 μl antibody diluent, 100 μl color developing solution, 100 μl stop solution and elution solution.

[0028] In the above-mentioned Tembusu virus antibody indirect ELISA diagnostic kit, the dilution ratio of the HRP-labeled goat anti-duck secondary antibody is 1:2000; the color developing solution is 50 mg TMB, 10 mL DMSO, 9.8 g citric acid and 1.2 mL 30% H2O2 added with distilled water to make up to 1000 mL; the stop solution is a mixture of 178.3 mL double distilled water and 21.7 mL 98% concentrated sulfuric acid.

[0029] The coating concentration of the duck Tembusu virus EDIII protein coating antigen expressed by yeast in the above-mentioned Tembusu virus antibody indirect ELISA diagnostic kit is 4 μg / mL.

[0030] Beneficial Effects

[0031] The present invention fully considers the codon preference of Pichia pastoris and the secondary structure of duck Tembusu virus EDIII, and uses the resistance G418 carried by the pPIC9K vector to screen the high copy of the target gene. Through these measures, the expression amount of EDIII protein after codon modification is greatly improved.

[0032] Secondly, the present invention uses methanol to induce expression and uses methanol as a carbon source, which solves the toxicity problem of adding IPTG to animals and the high cost problem of other prokaryotic expressions. At the same time, considering the disadvantage of difficult protein purification, the secretory expression of the genetically engineered bacteria of the present invention can produce eukaryotic protein products with natural biological activity, without the need for renaturation, and the purification process is simple, which can avoid the protein from existing in the form of inclusion bodies, and save the cumbersome steps of protein purification and renaturation.

[0033] In summary, the duck Tembusu virus EDIII gene was expressed in yeast, and was identified to have good antigenicity by SDS-PAGE and Western blotting tests, and also had the advantages of low production cost, simple purification method, and suitability for large-scale production. The yeast-expressed duck Tembusu virus EDIII protein obtained by the present invention is used as a coating antigen in an indirect ELISA diagnostic kit for Tembusu virus antibodies, which can improve the specificity, repeatability and stability of the kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the SDS-PAGE electrophoresis of the recombinant duck Tembusu virus EDIII after codon optimization; M is a protein marker; Well 1 is the culture supernatant harvested after 24 hours of ppIC9K-EDIII recombinant Pichia pastoris; Well 2 is the culture supernatant harvested after 48 hours of ppIC9K-EDIII recombinant Pichia pastoris; Well 3 is the culture supernatant harvested after 72 hours of ppIC9K-EDIII recombinant Pichia pastoris; Well 4 is the culture supernatant harvested after 96 hours of ppIC9K-EDIII recombinant Pichia pastoris.

[0035] Figure 2 This is the Western blotting image of the recombinant duck Tembusu virus EDIII after codon optimization. M is a protein marker; 1 and 2 are the reactions of EDIII protein with duck Tembusu virus positive serum. DETAILED DESCRIPTION

[0036] In order to make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Specific experimental methods not mentioned in the following examples are usually carried out according to conventional experimental methods.

[0037] Example 1 Yeast expression of duck Tembusu virus EDIII protein comprises the following steps:

[0038] 1. Optimization and acquisition of EDIII gene sequence

[0039] According to the codon preference of yeast, the duck Tembusu virus EDIII full gene is optimized and artificially synthesized. The present invention uses the method of full gene synthesis to use the codons TTG (Leu), AAG (Lys), TTC (Phe), CCA (Pro), TCT (Ser), GCT (Ala), GAC (Asp), AGA (Arg), AAC (Asn), TGT (Cys), CAA (Gln), GGT (Gly), CAC (His), ATC (Ile), ACT (Thr), TAC (Tyr), GTT (Val) preferred by Pichia pastoris to optimize and replace rare codons, and the expression amount is improved. In order to facilitate purification, 6 histidines (6×His tag) are added to the C-terminus, and EcoRⅠ and NotⅠ restriction endonuclease sites are introduced at both ends of the sequence. The optimized EDIII gene sequence is synthesized by Nanjing GenScript Biotechnology Co., Ltd., and its nucleotide sequence is shown in SEQ ID NO1.

[0040] 2. Construction of expression vector

[0041] The synthetic duck Tembusu virus EDIII gene and yeast vector ppIC9K were double-digested with two restriction endonucleases, EcoRⅠ and NotⅠ. The digested products were subjected to agarose gel electrophoresis and the DNA fragments were recovered using a gel recovery kit.

[0042] Add 6 μL of the target fragment after double enzyme digestion, 2 μL of the double enzyme digested plasmid, 1 μL of T4 DNA ligase, and 1 μL of ligase buffer to a sterile Eppendorf tube, mix thoroughly, and connect overnight at 4°C; add 10 μL of the ligation product to the competent E. coli DH5α, flick to mix, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, add 900 μL of LB liquid culture medium, shake and culture at 37°C on a shaker for 1 hour, spread the bacterial liquid on the LB solid culture medium containing ampicillin + kanamycin resistance, and culture at 37°C overnight. Pick a single colony from the transformed plate and inoculate it into 5 mL LB liquid culture medium containing ampicillin + kanamycin resistance, culture overnight at 37°C, and extract the plasmid with a plasmid extraction kit. The ligation product was double-digested with EcoRⅠ and NotⅠ, and sent to Bioengineering for sequencing analysis. The positive recombinant plasmid obtained was named ppIC9K-EDIII.

[0043] 3. Expression of recombinant protein

[0044] 1) Low-dose induction of expression

[0045] 5-10 μg of recombinant plasmid ppIC9K-EDIII was linearized using restriction endonuclease SalⅠ, and the linearized ppIC9K-EDIII was recovered using a PCR product purification kit and added to 90 μl competent Pichia pastoris GS115, mixed, and electroporated after 5 min of ice bath. After adding 1 mL of sorbitol, the mixture was placed at 30°C for 2 hours, and the bacterial solution was spread on ampicillin-resistant MD solid medium and cultured at 30°C for 3-4 days. A single colony was picked from the transformed plate and inoculated into 5 mL of YPD liquid medium containing ampicillin, cultured at 30°C and 220 rpm for 24 hours, centrifuged at 5000 rpm, and the supernatant was discarded and 10 mL of BMMY liquid medium was added. The mixture was cultured at 30°C and 250 rpm for 5 days, and methanol was added every 24 hours to maintain the methanol concentration at 1%. The supernatant was collected after centrifugation at 5000 rpm and analyzed by SDS-PAGE.

[0046] 2) High-dose induction of expression

[0047] The recombinant yeast strain GS115 / pPIC9K-EDIII was cultured in BMGY medium until OD600 was 3, and then centrifuged at 5000rpm for 15min to collect the cells, transferred to a conical flask, diluted with BMMY medium to OD600 of 1, sealed with 8 layers of sterile gauze, and cultured in a shaking incubator at 30°C and 220rpm for 96h. In order to maintain induced expression, 100% methanol was added every 24h to a final concentration of 1%. 1ml of the culture was taken into a sterile centrifuge tube at 24h, 48h, 72h, and 96h, respectively, and centrifuged at 12000rpm for 15min. The supernatant was collected and filtered through a 0.45μm filter membrane. After filtration, SDS-PAGE electrophoresis was performed. The results are shown in Figure 1 . M is a protein marker; Well 1 is the culture supernatant harvested after 24 hours of ppIC9K-EDIII recombinant Pichia pastoris; Well 2 is the culture supernatant harvested after 48 hours of ppIC9K-EDIII recombinant Pichia pastoris; Well 3 is the culture supernatant harvested after 72 hours of ppIC9K-EDIII recombinant Pichia pastoris; Well 4 is the culture supernatant harvested after 96 hours of ppIC9K-EDIII recombinant Pichia pastoris. As can be seen from the figure, the size of duck Tembusu virus EDIII protein is consistent with the predicted protein. Western blot identification showed that the duck Tembusu virus EDIII protein expressed in the yeast system can react with Tembusu virus positive serum. The results are shown in Figure 2 .

[0048] 4. Purification of recombinant protein using Ni-NTA Resin column

[0049] Add 50mM sodium phosphate, 0.3M sodium chloride, 10mM imidazole and 10mM pH8.0 Tris-HCl to the supernatant of induced expression, and filter with a 0.45μm filter. Take 10mL Ni-NTA Resin column and install it into XK16 / 20 chromatography column. After the column is installed, connect it to a constant flow pump, balance the column with 10 volumes of Buffer, load the sample after balancing, rinse the column with 8 volumes of Buffer after loading, and then elute the target protein with 8 volumes of BufferⅡ and collect it in 8 different tubes. The flow rate of the whole process is 5mL / min. The concentration of recombinant EDIII protein was determined by Bradford method to be 200ug / mL.

[0050] Example 2

[0051] The application of duck Tembusu virus EDIII protein expressed in yeast in the Tembusu virus antibody indirect ELISA diagnostic kit is as follows:

[0052] 1) Determination of the optimal coating concentration of antigen and the optimal dilution of serum

[0053] The matrix method was used to dilute the purified recombinant duck Tembusu virus EDIII protein with coating solution, and the dilution concentrations were set to 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, and 0.5μg / mL, respectively. 100μl / well was added to the ELISA plate from left to right and coated at 4°C overnight. The negative and positive sera were diluted with blocking solution, and the dilution ratios were 1:200, 1:400, 1:800, and 1:1600, respectively. 100μL / well was added to the ELISA plate coated with antigen from top to bottom, placed at 37°C for 1 hour, washed 3 times with PBST, 100μl HRP-labeled goat anti-duck secondary antibody was added to each well, reacted at 37°C for 1 hour, washed 3 times with PBST, 100μl substrate colorimetric solution was added to each well, color was developed for 10 minutes in the dark, 50μl stop solution was added to each well, and the OD value was read at 450nm on an ELISA reader. The results established that the optimal coating concentration of the coating antigen was 4 μg / mL, and the optimal serum dilution was 1:400;

[0054] 2) Assembly and optimization of the kit: The purified yeast-expressed EDIII protein was diluted to 4 μg / mL and added to the ELISA plate, 100 μL per well. The blocking solution was divided into three groups: 1% bovine serum albumin (BSA), 5% skim milk and 1% gelatin. The plate was blocked at 37°C for 2 h, and the optimal dilution of serum was added. The plate was repeated twice. Other conditions remained unchanged. The results showed that 5% skim milk had the best effect. The plate was divided into four groups according to the primary antibody incubation time, which were 30 min, 60 min, 90 min and 120 min. The plate was repeated twice. Other conditions remained unchanged. The results showed that the optimal binding time of the antigen and antibody was 60 min. The remaining conditions were that the dilution of the secondary antibody was 1:2000, the reaction conditions were 37°C, and the reaction time was 90 min.

[0055] 3) Determination of positive and negative critical values ​​of indirect ELISA method

[0056] The established indirect ELISA method was used to detect negative duck serum, and the average value (X) of OD450 of 30 Tembusu virus-negative sera was calculated to be 0.112, and the standard deviation (SD) was 0.062. The positive and negative critical value of the indirect ELISA detection method was determined to be 0.298 according to the formula: X+3SD, that is, when the OD450 value of the sample>0.298, it was judged to be positive. The critical value of ELSA negative was determined to be X+2SD=0.236, that is, when the OD450 value of the sample<0.236, it was judged to be negative, and 0.236<OD450 value of the sample to be tested<0.298 was suspicious.

[0057] Example 3: Detection of serum samples using the indirect ELISA diagnostic kit for duck Tembusu virus EDIII antibody of the present invention

[0058] (1) Dilute the yeast-expressed EDIII protein to 4 μg / mL and add 100 μl to each well of the ELISA plate. Coat the plate overnight at 4°C. Wash the plate three times with PBST, add 5% skim milk to each well, and block the plate at 37°C for 1 hour.

[0059] (2) Dilute the serum sample to be tested 400 times with PBST and add it to the ELISA plate, 100 μl per well, set up negative and positive controls, react at 37°C for 1 hour, and set up two replicates for all samples;

[0060] (3) Wash 3 times with PBST, add 100 μl 1:2000 HRP-labeled goat anti-duck secondary antibody to each well, react at 37°C for 90 min, wash 3 times with PBST, add 100 μl substrate colorimetric solution to each well, color for 8 min in dark, add 50 μl stop solution to each well, read OD value at 450 nm, repeat the experiment twice, the results are shown in Table 1:

[0061] Table 1: Average OD values ​​of 48 serum samples tested using the duck Tembusu virus antibody indirect ELISA diagnostic kit

[0062]

[0063] In Table 1, * is the average value of the negative control, and ** is the average value of the positive control.

[0064] According to the test kit criteria, the positive rate of duck Tembusu virus antibody was 54.3%. The diagnostic reagent had good inter-batch and intra-batch repeatability.

[0065] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a recombinant duck Tembusu virus E protein domain III protein, characterized in that: The steps include: (1) Construction of expression vector: The codon-optimized duck Tembusu virus EDIII gene shown in SEQ ID NO.1 was artificially synthesized, and restriction endonuclease cleavage sites were designed on the gene sequence. The EDIII gene sequence and the yeast expression vector pPIC9K were digested with the same restriction endonuclease and then connected together. The clones containing the EDIII gene sequence were detected and screened by double enzyme digestion; (2) Construction of yeast expression system: Use restriction endonuclease to linearize the recombinant expression plasmid and use electroporation to transform the plasmid. The gene sequence of EDIII was integrated into the genome of yeast GS115; the transformed bacterial suspension was spread on MD plates and cultured at 30°C; single colonies were picked for G418 resistance screening and PCR colony identification, and the obtained single colonies were induced to express at a small dose, and the expression supernatant was detected by SDS-PAGE to confirm the positive clones; (3) Purification and activity detection of recombinant proteins: Select positive clones for high-dose induced expression, and collect the supernatant from the expression The purified recombinant protein was purified using Protein Pure Ni-NTA resin and detected by SDS-PAGE and Western blot; Among them, the recombinant yeast is Mut + Phenotyping of recombinant yeast; The method for inducing expression of the recombinant yeast strain by fermentation and high-dose culture is as follows: the recombinant yeast strain GS115 / pPIC9K-EDIII is cultured in BMGY medium at 30°C and 220rpm for 24 hours, centrifuged, and 10mL of BMMY liquid medium is added after discarding the supernatant, and cultured at 30°C and 250rpm for 5 days, and methanol is supplemented every 24 hours to maintain the methanol concentration at 1%; or the recombinant yeast strain GS115 / pPIC9K-EDIII is cultured in BMGY medium until OD600 is 3, and the cells are collected by centrifugation, transferred to a conical flask, and BMMY medium is added to dilute to OD600 of 1, sealed with 8 layers of sterile gauze, and cultured in a shaker at 30°C and 220rpm for 96 hours, and 100% methanol is added every 24 hours to a final concentration of 1%.

2. A recombinant yeast expression plasmid for highly expressing duck Tembusu virus EDⅢ protein in yeast, characterized in that The recombinant yeast expression plasmid is a yeast expression vector pPIC9K in which the codon-optimized duck Tembusu virus EDⅢ gene shown in the artificially synthesized SEQ ID NO.1 is serially inserted into the Eco R Ⅰ and Not Ⅰ Obtained between two restriction endonuclease sites.

3. A recombinant yeast strain for highly expressing duck Tembusu virus EDⅢ protein in yeast, characterized in that: The recombinant yeast strain is obtained by using yeast cell GS115 as a host cell and transfecting the recombinant yeast expression plasmid described in claim 2.

4. Use of the recombinant expression plasmid according to claim 2 and the recombinant yeast strain according to claim 3 in the efficient expression and preparation of duck Tembusu virus EDⅢ protein.

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