A codon-optimized bovine viral diarrhea virus type 1 e2 protein gene and use thereof

Abstract

The embodiment of the present application relates to a codon-optimized bovine viral diarrhea virus type 1 E2 protein gene and application thereof, and belongs to the field of bioengineering, wherein the nucleotide sequence of the codon-optimized bovine viral diarrhea virus type 1 E2 protein gene is shown as SEQ ID NO:1. A CHO cell strain stably expressing bovine viral diarrhea virus E2 protein is successfully constructed, and the E2 protein expressed by the cell strain has the characteristics of high expression amount, good immunogenicity, easy purification and production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of veterinary biological products, in particular a codon-optimized bovine viral diarrhea virus type 1 E2 protein gene, a nucleic acid construct, a recombinant expression vector, an expression system cell, a preparation method of bovine viral diarrhea virus type 1 E2 protein, bovine viral diarrhea virus type 1 E2 protein, an immunogenic composition, a preparation method of a bovine viral diarrhea virus subunit vaccine, and their applications in preparing antigens, antibodies, subunit vaccines or detection kits of bovine viral diarrhea virus. BACKGROUND

[0002] Bovine viral diarrhea (BVD) is also known as mucosal disease, which is a contagious disease caused by bovine viral diarrhea virus (BVDV) with symptoms of diarrhea, fever, leukopenia, oral and digestive tract mucosal necrosis, pregnant cow abortion and fetal malformation. It is widely distributed worldwide and can persist and spread in cattle herds, seriously affecting the production performance of cattle, leading to decreased milk production, decreased milk quality, growth retardation, and developmental disorders. The disease mainly infects cattle, and calves are more susceptible, and it can also infect sheep, goats, pigs, deer and other ruminants. When pregnant cows are acutely infected, BVDV can be transmitted to the fetus through the placenta, causing fetal immune tolerance and becoming a persistently infected (PI) cow, which is the main source of BVDV transmission. The virus was first discovered in New York in 1946 from a cow with diarrhea, and was successfully isolated in 1957. It is now distributed worldwide, widely distributed in Europe, the Americas, Oceania, Asia, Africa and other countries and regions. The first strain of bovine viral diarrhea virus was isolated from a fetus aborted in China in 1980, and since then, reports of BVDV have increased, and the disease is now prevalent worldwide, causing significant losses to the cattle industry.

[0003] BVDV belongs to the Flaviviridae family and the Pestivirus genus, which also includes porcine pestivirus and border disease virus of sheep. BVDV is an enveloped virus with a particle size of about 40-60 nm, a single-stranded positive-sense RNA genome with a total length of about 12.3-12.5 kb, containing a 5' non-coding region, a large open reading frame, and a 3' non-coding region without a polyA tail. It encodes four structural proteins (C, E0, E1, E2) and eight non-structural proteins (NS1, NS2, NS2A, NS2B, NS3, NS4A, NS4B, NS5). proBVDV genome contains 5'NTR, C, Ems, E2, NS2, NS3, NS4A, NS4B, NS5A, NS5B). According to the difference of BVDV genome 5'NTR region, it can be divided into two genotypes, namely BVDV-1 and BVDV-2, and BVDV-3 has been reported in recent years. According to the pathogenicity of cell culture, it can be divided into two biotypes, namely cytopathogenic and non-cytopathogenic. E2 protein is an important structural protein of BVDV, which contains the main antigenic determinant of BVDV, can induce the production of neutralizing antibodies, can effectively neutralize the virus and inhibit the infection of BVDV, and is the first choice for the development of subunit vaccine. In addition, E2 protein has multiple neutralization sites, which is very important for virus RNA assembly or virus particle assembly, and the interaction between virus and receptor or infected cells.

[0004] Vaccine is the most effective tool for preventing infection, and several countries in the European Union have adopted measures such as eliminating persistent infection animals, improving vaccine protection, and strict biosecurity operations to purify bovine viral diarrhea. The effect is more obvious. At present, bovine viral diarrhea virus mainly uses inactivated vaccine and attenuated vaccine. Attenuated vaccine can quickly induce strong and long-lasting cellular and humoral immune response, but there is a risk of immunosuppression and bovine fetal pathogenicity. From the safety point of view, the cattle industry uses inactivated vaccine for prevention. But the protective effect of inactivated vaccine on cows and calves is poor, and the production of protective antibodies is slow and the side effects are large. At present, there is no vaccine that can effectively control the spread of BVDV and express high yield suitable for large-scale production. Therefore, it is still necessary to develop a bovine viral diarrhea vaccine with good immunogenicity, clinical safety, good protection, high yield and suitable for large-scale production.

[0005] The information disclosed in this Background section is only for the purpose of increasing the understanding of the general background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is already known to a person of ordinary skill in the art. SUMMARY

[0006] Invention objectives

[0007] The purpose of the present application is to provide a codon-optimized bovine viral diarrhea virus type 1 E2 protein gene, a nucleic acid construct, a recombinant expression vector, an expression system cell, a method for preparing bovine viral diarrhea virus type 1 E2 protein, bovine viral diarrhea virus type 1 E2 protein, immunogenic composition, method for preparing bovine viral diarrhea virus subunit vaccine, and their application in preparing bovine viral diarrhea virus antigen, antibody, subunit vaccine or detection kit.

[0008] The application successfully constructs a CHO cell strain stably expressing bovine viral diarrhea virus E2 protein, the E2 protein expressed by the cell strain has the characteristics of high expression amount, good immunogenicity, easy purification and production, and provides a technical means for better preventing and controlling the development of BVD.

[0009] Solution

[0010] To achieve the object of the present application, the following technical solutions are provided:

[0011] In one aspect, the present application provides a codon-optimized bovine viral diarrhea virus type 1 E2 protein gene, the nucleotide sequence of the codon-optimized bovine viral diarrhea virus type 1 E2 protein gene is shown as SEQ ID NO: 1.

[0012] The sequence of SEQ ID NO. 1 is the gene sequence of the optimized bovine viral diarrhea virus type 1 E2 protein gene:

[0013]

[0014] The gene sequence before optimization is shown in SEQ ID NO. 2 (the underlined part is the sequence removed after codon optimization):

[0015]

[0016] In a second aspect, a nucleic acid construct is provided, which comprises at least one copy of the codon-optimized BVDV-1 E2 protein gene of the first aspect.

[0017] Optionally, the nucleic acid construct further comprises a gene sequence encoding a signal peptide at the 5' end of the BVDV-1 E2 protein gene and / or a Kozak sequence; optionally, the nucleic acid construct further comprises a Kozak sequence and a gene sequence encoding a signal peptide at the 5' end of the BVDV-1 E2 protein gene, which contains a sequence as shown in SEQ ID NO: 3.

[0018] The sequence shown in SEQ ID NO: 3 is as follows:

[0019] gccaccatggagaccgataccctgctgctgtgggtgctgctgctgtgggtccctggctccaccggc.

[0020] Optionally, the nucleic acid construct further comprises a gene sequence encoding a tag at the 3' end of the BVDV-1 E2 protein gene; optionally, the tag is selected from at least one of a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and a SUMO tag; preferably, the tag is a His tag (the sequence of the His tag is shown in SEQ ID NO: 5: caccatcaccatcatcac ).

[0021] Optionally, the nucleic acid construct has a nucleotide sequence as shown in SEQ ID NO: 4:

[0022]

[0023] In a third aspect, a recombinant expression vector of the nucleic acid construct of the second aspect is provided; preferably, the recombinant expression vector adopts a pCEP4 vector.

[0024] In a fourth aspect, an expression system cell of the recombinant vector of the third aspect is provided; preferably, the expression system cell is a mammalian cell; further preferably, the mammalian cell is a CHO-B10 cell (referring to a CHO cell containing the recombinant vector of the third aspect (referred to as B10)).

[0025] In a fifth aspect, a method for preparing a BVDV-1 E2 protein is provided, which comprises the following steps: using the expression system cell of the fourth aspect to perform expression, collecting the cell supernatant after expression, and purifying to obtain the BVDV-1 E2 protein.

[0026] In a sixth aspect, there is provided a BVDV-1 E2 protein having immunogenicity, optionally, the amino acid sequence of which is shown as SEQ ID NO: 7, optionally, the BVDV-1 E2 protein is prepared by the method of the fifth aspect.

[0027] The amino acid sequence of SEQ ID NO: 7 is encoded by the sequence of SEQ ID NO: 1, and the amino acid sequence of SEQ ID NO: 7 is as follows:

[0028] HLDCKPEYSYAIAKSDRIGLQGAEDLTTVWKDYSHGMTLEDTMVIAWCKDGKLTYYARCTRETRYLAILHSRALPTSVVFKKLFEGQGQEDTVEMDDNFEFGLCPCDAKPIVRGTYNTTLLNGPAFQMVCPIGWTGTVSCMLANRDTLDTAVVRTYRRSRPFPYRQGCITQKTLGEDLYDCILGGNWTCVTGDQLQYTGGSVKSCKWCGFKFKKSEGLPHYPIGKCRLKNETGYRFVDGTSCNREGVAIVPQGLVKCKIGDTIVQVIALDTKLGPMPCKPYEIIPSEGPVEKTACTFNYTRTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYFAESI.

[0029] In a seventh aspect, there is provided an immunogenic composition comprising the codon-optimized BVDV-1 E2 protein gene of the first aspect, or the nucleic acid construct of the second aspect, or the recombinant expression vector of the third aspect, or the cell of the expression system of the fourth aspect, or the BVDV-1 E2 protein prepared by the method of the fifth aspect, or the BVDV-1 E2 protein having immunogenicity of the sixth aspect, and a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

[0030] Optionally, the adjuvant comprises 605 adjuvant, and the weight ratio of the purified BVDV-1 E2 protein to the 605 adjuvant is 1: (0.8-1.5).

[0031] Further, the immunogenic composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation.

[0032] Preferably, the nasal spray is selected from the group consisting of an aerosol, a spray and a powder spray.

[0033] Preferably, the oral formulation is selected from the group consisting of tablets, powders, pills, granules, granulates, soft / hard capsules, film-coated tablets, pellets, sublingual tablets and pastes.

[0034] Preferably, the parenteral formulation is a transdermal agent, an ointment, a plaster, a liquid for external use, an injectable or a bolus formulation.

[0035] In an eighth aspect, there is provided a method for preparing a bovine viral diarrhea virus subunit vaccine, comprising adding a pharmaceutically acceptable vaccine adjuvant to the bovine viral diarrhea virus type 1 E2 protein prepared by the method of the fifth aspect or the immunogenic bovine viral diarrhea virus type 1 E2 protein of the sixth aspect,

[0036] Optionally, the adjuvant comprises 605 adjuvant, and the weight ratio of the purified bovine viral diarrhea virus type 1 E2 protein to the 605 adjuvant is 1:(0.8-1.5).

[0037] In a ninth aspect, the codon-optimized bovine viral diarrhea virus type 1 E2 protein gene of the first aspect, the nucleic acid construct of the second aspect, the recombinant expression vector of the third aspect, the cell of the expression system of the fourth aspect, the bovine viral diarrhea virus type 1 E2 protein prepared by the method of the fifth aspect, the immunogenic composition of the seventh aspect, or the bovine viral diarrhea virus subunit vaccine prepared by the method of the eighth aspect is used for preparing an antigen, an antibody or a subunit vaccine or a detection kit of bovine viral diarrhea virus.

[0038] Advantages

[0039] (1) The present application can efficiently and stably express bovine viral diarrhea virus type 1 E2 protein, prepare a vaccine preparation for preventing bovine viral diarrhea type 1 disease, and reduce the loss caused by bovine viral diarrhea type 1 disease (BVDV-1) to the cattle industry.

[0040] (2) The present application modifies and optimizes the E2 protein gene sequence to make the immunogenicity of the protein better. By mixing the vaccine with the 605 adjuvant of the patent, the immunization effect of the vaccine is further enhanced. BRIEF DESCRIPTION OF DRAWINGS

[0041] One or more embodiments are illustrated by way of example in the figures that form a part of this patent document. This illustration is not to be considered limiting in relation to the embodiments illustrated and / or described in this patent document. The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0042] Figure 1Figure 1 is the result of double enzyme digestion electrophoretogram of the expression vector pCEP4-E2 (optimized) in Example 1 of the present application; M is DL10000 marker; 1 is the double enzyme-digested pCEP4-E2 (optimized) ;

[0043] Figure 2 Figure 1 is the result of double enzyme digestion electrophoretogram of the expression vector pCEP4-E2 (optimized) in Example 1 of the present application; M is DL10000 marker; 1 is the double enzyme-digested pCEP4-E2 (optimized) ;

[0044] Figure 3 Figure 1 is the result of double enzyme digestion electrophoretogram of the expression vector pCEP4-E2 (optimized) in Example 1 of the present application; M is DL10000 marker; 1 is the double enzyme-digested pCEP4-E2 (optimized) ; DETAILED DESCRIPTION

[0045] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0046] In addition, in order to better illustrate the present application, numerous specific details are given in the following detailed description. Those skilled in the art should understand that the present application can be implemented without some specific details. In some embodiments, the raw materials, elements, methods, means and the like which are well known to those skilled in the art are not described in detail, so as to highlight the main idea of the present application.

[0047] Unless otherwise explicitly indicated, in the entire specification and claims, the term "comprise" or its variants such as "contain" or "include" and the like will be understood to include the stated element or component, but not to exclude other elements or components.

[0048] The plasmids and bacterial strains involved in the present application are commercially available products.

[0049] In the following embodiments, the 605 adjuvant is a patent adjuvant (patent publication number CN103083659A) applied by Beijing Huaxia Xingyang Biological Technology Co., Ltd., and has been applied to the research and production of vaccines. The new veterinary drug products of Mannheimia haemolytica inactivated vaccine and infectious bovine rhinotracheitis inactivated vaccine developed by using the 605 adjuvant have obtained the national new veterinary drug registration certificate.

[0050] In the following embodiment, the BVDV-1 SD virus strain is isolated by Beijing Shengtail Technology Co., Ltd., and is a conventional BVDV-1 virus, which can be purchased by the general public through the public channel of China Veterinary Microbial Culture Collection Management Center.

[0051] The plasmid pCEP4 is purchased from Wuhan Moliang Biotechnology Co., Ltd.

[0052] The CHO cell involved in the present application is purchased from Merck.

[0053] The main reagents involved in the present application: the viral DNA / RNA extraction kit, one-step RT-PCR kit, DL2000 Marker, DL10000 Marker, and gel recovery kit are purchased from Beijing Quansijin Biotechnology Co., Ltd.; the endotoxin-removed plasmid extraction kit is purchased from QIAGEN Company; the endotoxin-removed plasmid extraction kit is purchased from Promega Company; Kpn I and Not I restriction endonucleases are purchased from takara Bioengineering (Dalian) Co., Ltd.; T4 DNA ligase is purchased from Beijing Quansijin Biotechnology Co., Ltd. LTX transfection reagent and hygromycin B are purchased from Thermo Fisher Scientific (China) Co., Ltd.; the bovine viral diarrhea antibody detection kit is purchased from IDEXX; the 605 adjuvant is provided by Beijing Shengtail Technology Co., Ltd.; the CD CHO medium is provided by Yishengke (Shenzhen) Co., Ltd.; and the stirring type bioreactor (5L) is purchased from Guangzhou QiZhi Biological Engineering Equipment Co., Ltd.

[0054] Example 1: Establishment of a CHO cell line stably expressing BVDV-1 E2 protein

[0055] 1.1 Synthesis of BVDV E2 protein gene

[0056] The E2 gene (hereinafter referred to as “E2 gene”) nucleotide sequence of the bovine viral diarrhea virus type 1 SD strain is codon-optimized, and the codon-optimized E2 gene sequence is shown in SEQ ID NO: 1, and the E2 gene sequence before codon optimization is shown in SEQ ID NO: 2.

[0057] The Kozak sequence (sequence marked with a wavy line) and the sequence encoding the signal peptide (bold sequence) at the 5' end of the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 are modified as shown in SEQ ID NO: 3, and the Kpn I enzyme cutting site (ggtacc) is modified. caccatcaccatcatcac ​), and a stop codon (tga) and a Not I restriction enzyme site (gcggccgc). The modified sequence of the codon-optimized E2 gene is shown in SEQ ID NO: 4, and the modified sequence of the E2 gene before codon optimization is shown in SEQ ID NO: 6. The sequences of SEQ ID NO: 4 and 6 were completed by General Biosystems (Anhui) Co., Ltd.

[0058] 1.2 Construction and identification of pCEP4-BVDV1-E2 recombinant transfer vector

[0059] The above-synthesized E2 genes before and after codon optimization (SEQ ID NO: 4, 6) and the pCEP4 vector were respectively double-digested with Kpn I and Not I, and the purified linear vector pCEP4 and the E2 genes before and after codon optimization were recovered by gel recovery, and were ligated using T4 DNA ligase, and the ligated recombinant plasmids were named pCEP4-E2 (optimized) and pCEP4-E2 (unoptimized). After ligation, E. coli DH5a competent cells were transformed, and were cultured at 37°C for 12-18 h. Single colonies on the culture plate were inoculated in LB medium containing ampicillin (Amp+), and were cultured at 37°C for 10-14 h, and the culture liquid was used as a template for bacterial liquid PCR identification, and positive clones were preliminarily screened. After the plasmid was extracted, pCEP4-E2 (optimized) and pCEP4-E2 (unoptimized) were double-digested with Kpn I and Not I, respectively, and the double-digested results of pCEP4-E2 (optimized) are shown in Figure 1 The positive bacterial liquid screened was sent to Shengong for sequencing, and the sequencing results were analyzed.

[0060] After the positive bacteria screened were cultured, plasmid DNA was extracted according to the Promega endotoxin-free plasmid extraction kit. The plasmid was linearized after enzyme digestion, and was recovered.

[0061] 1.3 Recombinant plasmid transfection of CHO cells and screening of CHO cells stably expressing BVDV-1 E2 protein

[0062] 1.3.2 Cell transfection and screening

[0063] Once the CHO cell density reached approximately 80%, the cells were transfected with linearized recombinant plasmids (pCEP4-E2 (optimized) and pCEP4-E2 (unoptimized)) according to the transfection reagent instructions. After 48 hours of culture, the cells were placed in selective medium containing hygromycin (200 μg / ml) for pressure selection culture. After 3-4 days, the cells were collected and passaged in 96-well plates using the limiting dilution method. Once resistant colonies reached sufficient size, single colonies were picked for expansion culture. Drug screening continued until stably expressing transgenic cell lines were obtained. CHO cells containing the optimized codon of the E2 gene were named CHO-B10 cells, and CHO cells containing the unoptimized codon of the E2 gene were named CHO-B10' cells.

[0064] 1.4 Cell shake-flask fermentation

[0065] CHO-B10 and CHO-B10' cells were respectively loaded with 5×10 5 Cells were seeded at 125 mL in a shake flask and cultured at 37 °C in a 5% CO2 constant temperature shaker. Samples were taken every 24 hours to monitor protein expression. After 11 days of culture, the cells were harvested.

[0066] 1.5 Protein Purification

[0067] Wash the packed Ni-NTA affinity resin with binding buffer for 10 column volumes and allow it to equilibrate. Add the sample to be purified to the equilibrated column. Wash the binding resin with 40 mM imidazole wash buffer (10 column volumes), then elute with 300 mM imidazole eluent (5-6 column volumes). Collect the eluent. The SDS-PAGE electrophoresis results of the CHO-B10 cell expression solution are shown below. Figure 2 As shown, the results indicate that this method can effectively obtain purified protein.

[0068] 1.6 Protein concentration and purity determination

[0069] The purity was detected by SDS-PAGE, and the purity of the optimized proteins was above 75%. The protein concentration was determined by BCA method. The protein concentrations before and after optimization are shown in Table 1, indicating that the protein expression level was significantly improved after codon optimization, by at least 3 to 5 times.

[0070] Table 1 Comparison of antigen expression levels before and after optimization (μg / ml)

[0071]

[0072] 1.7 CHO cell expression stability test

[0073] The codon-optimized CHO cells expressing bovine viral diarrhea E2 protein were routinely passaged to 20 generations, and F5, F10, F15 and F20 generation cells were taken for shake flask fermentation, and the protein concentration was detected. The results showed that the protein concentrations of F5, F10, F15 and F20 generation cells were 188 μg / ml, 192 μg / ml, 209 μg / ml and 189 μg / ml respectively. The CHO cells could still stably express after 20 generations of subculture.

[0074] Example 2: Preparation of vaccine and immunization test

[0075] 1.1 Preparation of bovine viral diarrhea virus type 1 E2 protein

[0076] When the density of CHO cells reached 2-3 x 106 cells / ml, the reactor process parameters were determined by adjusting pH and dissolved oxygen content for protein expression. The supernatant was collected by centrifugation at 6000-8000 r / min for 30 minutes, which was the protein antigen solution. The protein was purified by affinity chromatography method through NI-NTA purification column, and desalted by tangential flow filtration system after purification.

[0077] The type 1 E2 protein obtained by using the codon-optimized E2 protein gene was named "codon-optimized E2 protein".

[0078] The type 1 E2 protein obtained by using the E2 protein gene before codon optimization was named "E2 protein before codon optimization".

[0079] 1.2 Preparation of vaccine

[0080] The bovine viral diarrhea virus type 1 protein antigen that passed the test was mixed with 605 adjuvant at a ratio of 1:1, and nystatin solution was added at the same time to make the final concentration not more than 30 μg / ml. The mixture was stirred at 100-300 r / min until it was evenly mixed.

[0081] Two kinds of vaccines were obtained as follows:

[0082] The type 1 E2 protein obtained by using the codon-optimized E2 protein gene was named "codon-optimized E2 protein vaccine".

[0083] The type 1 E2 protein obtained by using the E2 protein gene before codon optimization was named "E2 protein before codon optimization vaccine".

[0084] Meanwhile, the control patent vaccine was prepared by the method in the prior art (patent application number: 201611208261.8).

[0085] 1.3 Evaluation of immunization effect

[0086] Use 2-6 months old healthy susceptible cattle (BVDV antigen antibody negative) 20 heads. Immunization group 3 groups, respectively, the codon optimization before the vaccine group (corresponding to the codon optimization before E2 protein vaccine), codon optimization after the vaccine group (corresponding to the codon optimization after E2 protein vaccine), and the control patent vaccine group (corresponding to the vaccine prepared by the method of patent application number: 201611208261.8), while setting up control group of 5 cattle (healthy susceptible cattle). Immunization group of cattle each neck muscle injection vaccine 2.0ml, immunization 21 days with the same method and dose of booster immunization 1 times, two immunization 14 days, together with 5 control cattle, blood sampling and using IDEXX kit to determine the antibody titer, at the same time, the protection test (attack virus using BVDV-1SD virus strain). Results as shown in Figure 3 As shown in Table 2, the antibody titer (antibody S / P value) and the protection rate of the attack virus of the present application group are higher than those of the control patent group, especially the protection rate of the attack virus is significantly better than that of the control patent group.

[0087] Table 2: Antibody titer before and after immunization of each group of cattle and protection rate after immunization

[0088]

[0089] The above results show that the protection rate of the attack virus after immunization of the vaccine prepared by the present application is better than that of the existing control patent vaccine group, although the codon optimization removes part of the amino acid sequence, but the immunogenicity is still maintained at a high level.

[0090] Finally, it should be noted that: the above examples are used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A nucleic acid construct comprising a codon-optimized bovine viral diarrhea virus type 1 E2 protein gene; The nucleotide sequence of the codon-optimized bovine viral diarrhea virus type 1 E2 protein gene is shown in SEQ ID NO:1; The nucleotide sequence of the nucleic acid construct is shown in SEQ ID NO:

4.

2. A recombinant expression vector containing the nucleic acid construct of claim 1.

3. The recombinant expression vector according to claim 2, characterized in that, The recombinant expression vector used is the pCEP4 vector.

4. An expression system cell comprising the recombinant expression vector of claim 2 or 3.

5. The expression system cell according to claim 4, characterized in that, The expression system cells are mammalian cells.

6. The expression system cell according to claim 5, characterized in that, The mammalian cells in question are CHO-B10 cells.

7. The use of the nucleic acid construct of claim 1, the recombinant expression vector of claim 2, or the expression system cell of any one of claims 4-6 in the preparation of a subunit vaccine for bovine viral diarrhea virus.

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