CN115894638A - Preparation method of recombinant duck tembusu virus E protein structural domain III protein - Google Patents
Preparation method of recombinant duck tembusu virus E protein structural domain III protein Download PDFInfo
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- CN115894638A CN115894638A CN202210987699.XA CN202210987699A CN115894638A CN 115894638 A CN115894638 A CN 115894638A CN 202210987699 A CN202210987699 A CN 202210987699A CN 115894638 A CN115894638 A CN 115894638A
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Abstract
The invention discloses a preparation method of a recombinant duck tembusu virus E protein structural domain III protein. The invention firstly provides a pichia pastoris GS115 strain for preparing a duck tembusu virus E protein structural domain III. The recombinant strain is obtained by intercepting an antigen region of duck tembusu virus envelope protein EDIII, obtaining a coding sequence of the duck tembusu virus EDIII through codon optimization, and then cloning the coding sequence to a yeast expression vector pPIC9K and transforming the coding sequence to a yeast host strain GS115. Under the action of methanol, the duck tembusu virus EDIII protein is efficiently expressed. Meanwhile, the protein is used as a coating antigen for a duck tembusu virus antibody indirect ELISA diagnostic kit, and the kit has high specificity, repeatability and stability and can be used for diagnosing related diseases caused by tembusu virus.
Description
Technical Field
The invention belongs to the field of veterinary biotechnology, and particularly relates to yeast expression and application of duck tembusu virus ED III protein.
Background
Duck Tembusu virus belonging to the family Flaviviridae, the genus Flaviviridae, the group of Entaya viruses. Tembusu virus was first isolated from aedes and culex mosquitos in malaysia and thailand. Although birds and poultry have been considered as natural vertebrate hosts of the virus, the reports of epidemic diseases of the birds and the poultry caused by the virus are few, and how waterfowls such as ducks and geese infect the disease is unknown and is estimated to be related to mosquitoes. Meanwhile, the tembusu virus can be detected from dead sparrows in a duck farm in a literature report, which indicates that the virus can be spread by birds, and the literature also indicates that the virus can be separated from cloaca of infected ducks, which indicates that the virus can be detoxified by excrement, pollute the environment, feed, drinking water, appliances and the like, and further cause the spreading of the virus.
The E protein is the main surface structural protein of the duck Tembusu virus, consists of 500 amino acid residues and comprises a plurality of antigen epitopes related to host tropism, host cell membrane fusion and host cell surface receptor binding. According to the function difference, the E protein is divided into three structural domains (I, II and III), and an immunoglobulin-like third structural domain (EDIII) is positioned at the outermost layer of the virus, has an important function in mediating the combination of the virus and a host receptor and is also a dominant epitope region for inducing a neutralizing antibody. Therefore, the method has important significance for the research on the expression and the function of the EDIII, the interaction of the virus and the host cell, clinical diagnosis and subunit vaccine research.
The establishment of the antibody detection method of the virus is of great significance to epidemiological investigation, immune effect evaluation and the like of the new epidemic disease, and ELISA is popular due to the characteristics of large detection flux, exact result, high sensitivity, easy basic popularization and the like, so that the method becomes the most widely applied serological detection method at present. Therefore, the duck tembusu virus EDIII gene is used as a template, the protein is obtained by a yeast expression method, and the purified recombinant EDIII protein is used as an antigen to establish an indirect ELISA detection method, so that a technical means is provided for early diagnosis and large-scale detection of the virus.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a yeast expression method of duck tembusu virus EDIII protein, which collects the duck tembusu virus EDIII protein as a coating antigen in a yeast expression mode, is used in a tembusu virus antibody indirect ELISA diagnostic kit, and improves the specificity, the repeatability and the stability of the kit.
The invention also aims to provide application of the yeast-expressed duck tembusu virus EDIII protein in a duck tembusu virus antibody indirect ELISA diagnostic kit.
The invention is realized by the following technical scheme:
the amino acid sequence of the duck tembusu virus EDIII protein is shown as SEQ ID NO. 2:
Met Gln Gly Leu Lys Leu Lys Gly Met Thr Tyr Pro Met Cys Ser Asn Thr Phe Ser Leu Val Lys Asn Pro Thr Asp Thr Gly His Gly Thr Val Val Val Glu Leu Ser Tyr Ala Gly Thr Asp Gly Pro Cys Arg Val Pro Ile Ser Met Ser Ala Asp Leu Asn Asp Met Thr Pro Val Gly Arg Leu Ile Thr Val Asn Pro Tyr Val Ser Thr Ser Ser Thr Gly Ala Lys Ile Met Val Glu Val Glu Pro Pro Phe Gly Asp Ser Phe Ile Leu Val Gly Ser Gly Lys Gly Gln Ile Arg Tyr Gln Trp His Arg Ser Gly Ser Thr Ile Gly Lys Ala Phe Thr Ser Thr Leu Lys Gly Ala His His His His
the invention further provides a preparation method of the recombinant duck tembusu virus EDIII protein, which comprises the following steps:
(1) Construction of the expression vector: artificially synthesizing a codon-optimized duck tembusu virus ED III gene shown in SEQ ID NO.1, designing a restriction enzyme cutting site on a gene sequence, connecting an EDIII gene sequence and a yeast expression vector pPIC9K together after being cut by the same restriction enzyme, and detecting and screening clone containing the EDIII gene sequence by double enzyme cutting;
(2) Construction of the Yeast expression System: integrating the gene sequence of EDIII into the genome of the yeast by an electrotransformation method by using a restriction endonuclease linearized recombinant expression plasmid to obtain a recombinant yeast strain GS115/pPIC9K-EDIII; coating the transformed thallus suspension on an MD (MD) flat plate, and culturing at 30 ℃; selecting a single colony, screening and identifying, and performing induced expression on the obtained single colony;
(3) And (4) purifying the recombinant protein.
As a preferable mode of the present invention, the preparation method comprises the following steps:
(1) Construction of the expression vector: designing restriction endonuclease sites on an artificially synthesized duck tembusu virus EDIII gene sequence, connecting the EDIII gene sequence and a yeast expression vector pPIC9K together after enzyme digestion by the same restriction endonuclease, and detecting and screening clones containing the EDIII gene sequence by double enzyme digestion.
(2) Construction of the Yeast expression System: integrating the gene sequence of the EDIII into the genome of the yeast by an electrotransformation method by using a restriction enzyme linearized recombinant expression plasmid; coating the transformed thallus suspension on an MD (MD) plate, and culturing at 30 ℃; and (3) selecting a single colony, performing G418 resistance screening and PCR colony identification, performing small-dose induced expression on the obtained single colony, and detecting an expression supernatant by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to confirm a positive clone.
(3) Purification and activity detection of recombinant proteins: selecting positive clone colonies for large-dose induced expression, purifying the expression supernatant by adopting Protein Pure Ni-NTA resin, and detecting the purified recombinant Protein by SDS-PAGE and Western blot.
As a preferable aspect of the present invention, the yeast cell in step (2) is GS115.
As a preferred embodiment of the present invention, the recombinant yeast strain GS115/pPIC9K-EDIII is a positive recombinant yeast with high copy number obtained by PCR identification and G418 resistant plate screening. The universal primers identified by PCR are 5'AOX1 and 3' AOX1, and the sequences are 5'-GACTGGTTCCAATTGACAAGC-3' and 5 '-GCAAATGGCATTCGACATCC-3'.
Preferably, the recombinant yeast is Mut + A phenotypic recombinant yeast.
As a preferred method of the invention, the method for fermenting and culturing the recombinant yeast strain comprises the following steps: culturing the recombinant yeast strain GS115/pPIC9K-EDIII in BMGY culture medium at 30 ℃ and 220rpm for 24 hours, centrifuging, removing supernatant, adding 10mL of BMMY liquid culture medium, culturing at 30 ℃ and 250rpm for 5 days, supplementing methanol every 24 hours, and maintaining the methanol concentration at 1%; or culturing the recombinant yeast strain GS115/pPIC9K-EDIII in a BMGY culture medium until OD600 is 3, centrifuging to collect the thallus, transferring to a triangular conical flask, adding the BMMY culture medium to dilute until the OD600 is 1, sealing by 8 layers of sterile gauze, performing shake culture at 30 ℃ and 220rpm for 96h, and adding 100% methanol every 24h until the final concentration is 1%.
Preferably, the Ni-NTA Resin column is used for separating and purifying the recombinant duck tembusu virus E protein structural domain III protein
A recombinant yeast expression plasmid for highly expressing duck tembusu virus ED III protein in yeast is obtained by connecting a codon-optimized duck tembusu virus ED III gene shown in an artificially synthesized SEQ ID NO.1 in series between EcoR I and Not I restriction endonuclease sites in a yeast expression vector pPIC 9K.
A recombinant yeast strain for highly expressing duck tembusu virus ED III protein in yeast is obtained by transfecting a recombinant yeast expression plasmid provided by the invention with a yeast cell GS115 as a host cell.
The recombinant yeast expression plasmid and the recombinant yeast strain are applied to efficient expression preparation of duck tembusu virus ED III protein.
The yeast-expressed duck tembusu virus EDIII protein disclosed by the invention is used as an envelope antigen in an indirect tembusu virus antibody ELISA diagnostic kit.
The invention discloses a tembusu virus antibody indirect ELISA diagnostic kit, which comprises an ELISA plate coated by using a duck tembusu virus EDIII protein expressed by the method as an antigen, 100 mul of duck tembusu virus positive control serum, 100 mul of duck tembusu virus negative control serum, 100 mul of HRP-labeled goat anti-duck secondary antibody, 100 mul of antibody diluent, 100 mul of color developing solution, 100 mul of stop solution and eluent.
In the tembusu virus antibody indirect ELISA diagnostic kit, the dilution ratio of the HRP-labeled goat anti-duck secondary antibody is 1: 2000; the color former was 50mg TMB,10mL DMSO,9.8g citric acid and 1.2mL 30% 2 O 2 Adding distilled water to a constant volume of 1000mL; the stop solution is prepared by mixing 178.3mL of double distilled water and 21.7mL of 98% concentrated sulfuric acid.
The coating concentration of the yeast-expressed duck tembusu virus EDIII protein coating antigen in the tembusu virus antibody indirect ELISA diagnostic kit is 4 mug/mL.
Advantageous effects
The invention fully considers the codon preference of pichia pastoris and the secondary structure condition of duck tembusu virus EDIII, and applies resistance G418 carried by a pPIC9K carrier to carry out high copy screening of target genes. By the measures, the expression quantity of the codon-modified EDIII protein is greatly improved.
Secondly, methanol is used for induction expression, and methanol is used as a carbon source, so that the problems of toxicity and high cost of animals caused by adding IPTG in other prokaryotic expression are solved. Meanwhile, in consideration of the defect of difficult protein purification, the secretory expression of the genetically engineered bacteria can generate a eukaryotic protein product with natural biological activity, the renaturation is not needed, the purification process is simple, the existence of the protein in an inclusion body form can be avoided, and the complicated steps of protein purification renaturation and the like are omitted.
In conclusion, the duck tembusu virus EDIII gene is expressed in yeast, has good antigenicity as identified by SDS-PAGE and Western blotting tests, and has the advantages of low production cost, simple purification method, suitability for large-scale production and the like. The yeast-expressed duck tembusu virus EDIII protein obtained by the invention is used as a coating antigen in a tembusu virus antibody indirect ELISA diagnostic kit, and can improve the specificity, repeatability and stability of the kit.
Drawings
FIG. 1 is a SDS-PAGE electrophoresis of codon optimized recombinant duck tembusu virus EDIII; m is protein marker; the 1 st hole is culture supernatant obtained after 24 hours of ppIC9K-EDIII recombinant pichia pastoris; the 2 nd hole is culture supernatant obtained after 48 hours of ppIC9K-EDIII recombinant pichia pastoris; the 3 rd hole is culture supernatant obtained after 72 hours of ppIC9K-EDIII recombinant pichia pastoris; the 4 th hole is culture supernatant obtained after 96h of ppIC9K-EDIII recombinant pichia pastoris.
FIG. 2 is a Western blotting picture of recombinant duck tembusu virus EDIII after codon optimization. M is protein marker; 1. and 2, the EDIII protein reacts with duck tembusu virus positive serum.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention, and that specific experimental procedures not mentioned in the following examples are generally performed according to conventional experimental procedures.
Example 1 yeast expression of duck tembusu virus EDIII protein, comprising the steps of:
1. optimization and acquisition of EDIII Gene sequences
Optimizing and artificially synthesizing the duck Tembusu virus EDIII whole gene according to the codon preference of yeast. The invention optimizes and replaces rare codons by using 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) and GTT (Val) favored by pichia pastoris by using a whole-gene synthesis method, thereby improving the expression quantity. For ease of purification, 6 histidines (6 × His tag) were added at the C-terminus, and EcoRI and Not I restriction enzyme sites were introduced at both ends of the sequence. The optimized EDIII gene sequence is synthesized by Nanjing Kingsler Biotech Co., ltd, and the nucleotide sequence is shown as SEQ ID NO 1.
2. Construction of expression vectors
Carrying out double enzyme digestion on the synthesized duck tembusu virus EDIII gene and the yeast carrier ppiC9K by using two restriction endonucleases EcoR I and Not I, carrying out agarose gel electrophoresis on the product after enzyme digestion, and recovering a DNA fragment by using a gel recovery kit.
Respectively adding 6 mu L of double-enzyme-digested target fragment, 2 mu L of double-enzyme-digested plasmid, 1 mu L of T4DNA ligase and 1 mu L of ligase buffer solution into a sterile Eppendorf tube, fully and uniformly mixing, and connecting overnight at 4 ℃; adding 10 mu L of the ligation product into escherichia coli competent DH5 alpha, flicking and uniformly mixing, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 90s, adding 900 mu L of LB liquid culture medium, shaking and culturing for 1h at 37 ℃ on a shaking bed, coating the bacterial liquid on LB solid culture medium with ampicillin and kanamycin resistance, and culturing overnight at 37 ℃. A single colony was picked from the transformed plate and inoculated into 5mL LB liquid medium containing ampicillin + kanamycin resistance, cultured overnight at 37 ℃ and plasmid was extracted with a plasmid extraction kit. The ligation product was identified by double digestion with EcoR I and Not I, and simultaneously subjected to sequencing analysis, and the resulting positive recombinant plasmid was named as PPIC9K-EDIII.
3. Expression of recombinant proteins
1) Small dose induced expression
Linearizing 5-10 mu g of recombinant plasmid ppIC9K-EDIII by using restriction enzyme SalI, recovering the linearized ppIC9K-EDIII by using a PCR product purification kit, adding the linearized ppIC9K-EDIII into 90 mu l of competent pichia pastoris GS115, uniformly mixing, carrying out electric shock transformation after 5min in ice bath, adding 1mL of sorbitol, standing for 2 hours at 30 ℃, coating a bacterium solution on an ampicillin-resistant MD solid culture medium, and culturing for 3-4 days at 30 ℃. Single colonies were picked from the transformed plates and inoculated into 5mL YPD liquid medium containing benzyl amine, cultured at 30 ℃ for 24 hours at 220rpm, centrifuged at 5000rpm, the supernatant was discarded, 10mL BMMY liquid medium was added, cultured at 30 ℃ for 5 days at 250rpm, and supplemented with methanol at intervals of 24 hours, with the methanol concentration maintained at 1%. After centrifugation at 5000rpm, the supernatant was collected and analyzed by SDS-PAGE.
2) Large dose induced expression
And (3) culturing the recombinant yeast strain GS115/pPIC9K-EDIII in a BMGY culture medium until OD600 is 3, centrifuging at 5000rpm for 15min to collect thalli, transferring to a triangular conical flask, adding a BMMY culture medium to dilute until OD600 is 1, sealing with 8 layers of sterile gauze, and performing shake culture at 30 ℃ and 220rpm for 96h. To maintain the induced expression, 100% methanol was added every 24 hours to a final concentration of 1%, 1ml of the culture was taken in a sterilized centrifuge tube at 24 hours, 48 hours, 72 hours, and 96 hours, respectively, centrifuged at 12000rpm for 15min, and the supernatants were collected, filtered through a 0.45 μm filter, and subjected to SDS-PAGE, and the results are shown in FIG. 1.M is protein marker; the 1 st hole is culture supernatant obtained after 24h of ppIC9K-EDIII recombinant pichia pastoris; the 2 nd hole is culture supernatant obtained after 48h of ppIC9K-EDIII recombinant pichia pastoris; the 3 rd hole is culture supernatant obtained after 72h of ppIC9K-EDIII recombinant pichia pastoris; and the 4 th hole is culture supernatant which is harvested 96h after the ppIC9K-EDIII recombinant pichia pastoris. As can be seen from the figure, the size of the duck tembusu virus EDIII protein is consistent with the predicted protein. Western blot identification, the duck tembusu virus EDIII protein expressed by a yeast system can react with the tembusu virus positive serum, and the result is shown in figure 2.
4. Purification of recombinant protein Ni-NTA Resin column
To the supernatant of the induced expression, 50mM sodium phosphate, 0.3M sodium chloride, 10mM imidazole and 10mM Tris-HCl pH8.0 were added at the final concentration, and the mixture was filtered through a 0.45 μ M filter. And (2) filling a 10mL Ni-NTA Resin column into an XK16/20 chromatographic column, connecting the column with a constant flow pump after the column is filled, balancing the column by using a Buffer with 10 times volume, loading the sample after the column is balanced, washing the column by using a Buffer with 8 times volume after the loading is finished, eluting the target protein by using a Buffer II with 8 times volume, and collecting the target protein into 8 different tubes, wherein the flow rate of the whole process is 5mL/min. The concentration of recombinant EDIII protein was determined to be 200ug/mL by Bradford method.
Example 2
The yeast-expressed duck tembusu virus EDIII protein tembusu virus antibody indirect ELISA diagnostic kit comprises the following specific steps:
1) Determination of optimal coating concentration of antigen and optimal dilution of serum
And (3) diluting the purified recombinant duck tembusu virus EDIII protein by using a matrix method through a coating solution, setting the dilution concentrations to be 16 mu g/mL, 8 mu g/mL, 4 mu g/mL, 2 mu g/mL,1 mu g/mL and 0.5 mu g/mL respectively, adding 100 mu l/hole into an enzyme label plate from left to right, and coating overnight at 4 ℃. The negative serum and the positive serum are diluted by a blocking solution, the dilution ratio is respectively 1. The results establish that the optimal coating concentration of the coating antigen is 4. Mu.g/mL, and the optimal dilution factor of the serum is 1:400;
2) Assembling and optimizing the kit: diluting the purified yeast-expressed EDIII protein to 4 mug/mL, adding the diluted yeast-expressed EDIII protein to an enzyme label plate, wherein each hole is 100 mug L, the diluted yeast-expressed EDIII protein is divided into three groups, namely 1% Bovine Serum Albumin (BSA), 5% skim milk and 1% gelatin according to the types of sealing solutions, sealing the holes at 37 ℃ for 2h, adding serum with the optimal dilution degree, repeating the two holes, keeping other conditions unchanged, and displaying that the effect of the 5% skim milk is optimal according to the result; dividing the primary antibody into four groups according to the incubation time, namely 30min, 60min, 90min and 120min, repeating two holes, keeping other conditions unchanged, showing that the optimal combination time of the antigen and the antibody is 60min according to the result, and determining the rest conditions that the dilution of the secondary antibody is 1;
3) Determination of negative and positive critical value of indirect ELISA method
Negative duck sera were tested by the established indirect ELISA method and the mean (X) of the 30 tembusu virus negative sera OD450 was calculated to be 0.112 and the Standard Deviation (SD) 0.062, according to the formula: x +3SD determines that the negative-positive threshold of the indirect ELISA detection method is 0.298, i.e., positive when the OD450 value of the sample is >0.298, and determines that the negative threshold of the ELSA is X +2sd =0.236, i.e., negative when the OD450 value of the sample is <0.236, and 0.236< OD450 value of the sample to be tested <0.298 is suspicious.
Example 3 detection of serum Using the Indirect ELISA diagnostic kit for Duck Tembusu Virus EDIII antibody of the present invention
(1) Diluting the EDIII protein expressed by yeast to 4 mu g/mL, adding the diluted EDIII protein to an enzyme label plate, coating 100 mu l of the protein in each hole at 4 ℃ overnight, washing the plate for 3 times by PBST, adding 5% skim milk in each hole, and sealing for 1 hour at 37 ℃;
(2) Diluting a serum sample to be detected by 400 times by PBST, adding the diluted serum sample to an enzyme label plate, setting negative and positive controls in each hole by 100 mu l, reacting for 1 hour at 37 ℃, and setting two repeats for all samples;
(3) PBST was washed 3 times with 100 μ l 1:2000 HRP-labeled goat anti-duck secondary antibody, reacting at 37 ℃ for 90min, washing with PBST for 3 times, adding 100 ul of substrate color development liquid into each hole, developing for 8 minutes in a dark place, adding 50 ul of stop solution into each hole, reading OD value with a reading instrument at 450nm, repeating the experiment twice, and obtaining the following results in the following table 1:
table 1: detection of average OD value corresponding to 48 serum samples by using duck tembusu virus antibody indirect ELISA diagnostic kit
In table 1, is the average value of negative controls, and is the average value of positive controls.
According to the judgment standard of the kit, the duck tembusu virus antibody positive rate is 54.3%. The diagnostic reagents have good inter-batch and intra-batch repeatability.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (10)
1. A preparation method of a recombinant duck tembusu virus E protein structural domain III protein is characterized by comprising the following steps:
(1) Construction of the expression vector: artificially synthesizing a codon-optimized duck tembusu virus ED III gene shown in SEQ ID NO.1, designing restriction endonuclease restriction sites on a gene sequence, connecting an EDIII gene sequence and a yeast expression vector pPIC9K together after enzyme digestion by the same restriction endonuclease, and detecting and screening clone containing the EDIII gene sequence by double enzyme digestion;
(2) Construction of the Yeast expression System: integrating the gene sequence of EDIII into the genome of the yeast by an electrotransformation method by using a restriction endonuclease linearized recombinant expression plasmid to obtain a recombinant yeast strain GS115/pPIC9K-EDIII; coating the transformed thallus suspension on an MD (MD) flat plate, and culturing at 30 ℃; selecting a single colony, screening and identifying, and performing induced expression on the obtained single colony;
(3) And (4) purifying the recombinant protein.
2. The method according to claim 1, wherein the yeast cell of step (2) is GS115.
3. The method according to claim 1, wherein the recombinant yeast strain GS115/pPIC9K-EDIII is a positive recombinant yeast with high copy number obtained by PCR identification and G418 resistant plate screening.
4. A method according to claim 3, wherein the recombinant yeast is Mut + A phenotypic recombinant yeast.
5. The method according to claim 1, wherein the recombinant yeast strain is cultured by fermentation method: culturing the recombinant yeast strain GS115/pPIC9K-EDIII in BMGY culture medium at 30 ℃ and 220rpm for 24 hours, centrifuging, removing supernatant, adding 10mL BMMY liquid culture medium, culturing at 30 ℃ and 250rpm for 5 days, supplementing methanol every 24 hours, and maintaining the methanol concentration at 1%; or culturing the recombinant yeast strain GS115/pPIC9K-EDIII in a BMGY culture medium until OD600 is 3, centrifuging to collect the thallus, transferring to a triangular conical flask, adding the BMMY culture medium to dilute until the OD600 is 1, sealing by 8 layers of sterile gauze, performing shake culture at 30 ℃ and 220rpm for 96h, and adding 100% methanol every 24h until the final concentration is 1%.
6. The preparation method of claim 1, wherein the recombinant duck tembusu virus E protein domain III protein is separated and purified by using a Ni-NTA Resin column.
7. A recombinant yeast expression plasmid for highly expressing duck tembusu virus ED III protein in yeast is characterized in that the recombinant yeast expression plasmid is obtained by connecting a codon-optimized duck tembusu virus ED III gene shown in an artificially synthesized SEQ ID NO.1 in series between two restriction endonuclease sites EcoRI and Not I in a yeast expression vector pPIC 9K.
8. A recombinant yeast strain for highly expressing duck tembusu virus ED III protein in yeast, which is characterized in that the recombinant yeast strain is obtained by transfecting the recombinant yeast expression plasmid of claim 7 by taking a yeast cell GS115 as a host cell.
9. The recombinant expression plasmid of claim 7 and the recombinant yeast strain of claim 8 are applied to efficient expression and preparation of duck tembusu virus ED III protein.
10. Use of the recombinant duck tembusu virus ED III protein prepared by the preparation method according to any one of claims 1-6 as a coating antigen in preparation of a duck tembusu virus antibody indirect ELISA diagnostic kit.
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| CN118067991A (en) * | 2024-02-05 | 2024-05-24 | 北京亿森宝生物科技有限公司 | Duck tembusu virus detection kit and application thereof |
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