CN111808176B

CN111808176B - Bovine herpes virus antigen compositions and uses thereof

Info

Publication number: CN111808176B
Application number: CN202010891771.XA
Authority: CN
Inventors: 曹文龙; 孔迪; 滕小锘; 张大鹤
Original assignee: Suzhou Shinuo Biotechnology Co ltd
Current assignee: Suzhou Womei Biology Co ltd
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2020-12-04
Anticipated expiration: 2040-08-31
Also published as: CN111808176A

Abstract

The invention relates to a bovine herpes virus antigen composition and application thereof, wherein the bovine herpes virus antigen composition comprises at least two of the following proteins: a recombinant bovine herpes virus gB protein with an amino acid sequence shown as SEQ ID NO. 1, a recombinant bovine herpes virus gD protein with an amino acid sequence shown as SEQ ID NO. 2, a recombinant bovine herpes virus gH protein with an amino acid sequence shown as SEQ ID NO. 3 and a recombinant bovine herpes virus gL protein with an amino acid sequence shown as SEQ ID NO. 4. The sequence information and the spatial structure of the gB protein, the gD protein, the gH protein and the gL protein of the bovine herpes virus are comprehensively analyzed, and the four proteins are subjected to site-directed mutagenesis respectively, so that the respective immunogenicity of the proteins is improved, a neutralizing antibody can be better excited, the immune effect is better, and the immune period is longer.

Description

Bovine herpes virus antigen compositions and uses thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a bovine herpes virus antigen composition and application thereof.

Background

Infectious Bovine Rhinotracheitis (IBR) is a highly contagious disease caused by infection with Infectious Bovine Rhinotracheitis Virus (IBRV). The clinical symptoms caused by the disease mainly comprise dyspnea, rhinitis, fever, inappetence, conjunctivitis, mastitis, abortion, balanoposthitis, vulvovaginitis, systemic infection of the whole body and the like. IBRV can cause latent infection in dorsal ganglia and trigeminal ganglia, recessive infection can exist in infected cattle, and the infected cattle is ignored because of not showing any clinical characteristics, but the recessive infected cattle can continuously expel toxin to the outside, so that other cattle susceptible to infection are caused. The same is true for the generally recovered sick cattle, the virus is latent in the body, and the virus which stimulates the body is further activated when the external environment changes, so once the disease occurs, the disease is difficult to eliminate from the cattle herd, the prevalence and the harm are both increased and decreased, and huge economic losses are caused to the cattle industry. The world animal health Organization (OIE) defines the disease as a B-type epidemic disease, and the disease is classified as a second type of animal epidemic disease in the animal epidemic disease list by the Ministry of agriculture in China.

IBRV is also called Bovine herpes virus type I (BHV-1), and belongs to the family of herpesviridae, the subfamily of alpha herpes viruses. The IBRV is a linear double-stranded DNA virus with a capsule membrane, the size of the IBRV is about 135-140 kb, the diameter of the virus is 120-220 nm, the particle is spherical, and the capsid is in regular icosahedral symmetry. The IBRV genome consists of a long unique region (UL, 106 kb) and a short unique region (Us, 10 kb) and repetitive sequences IRs and TRs flanking the Us region. There are 33 structural proteins encoded by IBRV, 13 of which are associated with the nucleocapsid and 10 of which encode glycoproteins. Of these 10 glycoproteins, 6 are in the long unique sequence regions, designated gK (UL 53), gC (UL 44), gB (UL 27), gH (UL 22), gM (ULl 0) and gl (ull), respectively, and the remaining four are in the short unique sequence regions, designated gG (US 4), gD (US 6), gI (US 7) and gE (US 8). It is now known that at least 4 glycoproteins, namely the gB, gD, gH and gL proteins, are required for herpesvirus entry into cells. The gB protein is related to virus adsorption and penetration of host cells, can simultaneously induce an organism to generate humoral immunity and cellular immunity, and is one of main antigen proteins for inducing the organism to generate immune response. The gD protein is located on the surface of the virus envelope and infected cells, is necessary for virus replication, is related to virus penetration and entry into host cells, and can simultaneously induce the body to generate humoral immunity and cellular immunity. The heterodimer gH-gL formed by the gH protein and gL protein is essential in the invasion of herpesvirus into host cells, fusion of virus with cells or between cells, and spread of virus between cells.

At present, two modes of killing and vaccination are mainly adopted for controlling IBR globally, but killing cost is huge, and the problems of poor immune effect, short immune period and the like exist in common vaccines.

Disclosure of Invention

Based on the above, it is necessary to provide a bovine herpes virus antigen composition having a good immune effect and a long immune duration.

A bovine herpesvirus antigen composition comprising at least two of the following proteins: a recombinant bovine herpes virus gB protein with an amino acid sequence shown as SEQ ID NO. 1, a recombinant bovine herpes virus gD protein with an amino acid sequence shown as SEQ ID NO. 2, a recombinant bovine herpes virus gH protein with an amino acid sequence shown as SEQ ID NO. 3 and a recombinant bovine herpes virus gL protein with an amino acid sequence shown as SEQ ID NO. 4.

In one embodiment, the recombinant bovine herpes virus gB protein, the recombinant bovine herpes virus gD protein, the recombinant bovine herpes virus gH protein, and the recombinant bovine herpes virus gL protein are included.

In one embodiment, the recombinant bovine herpes virus gB protein and the recombinant bovine herpes virus gD protein form a heterodimer and the recombinant bovine herpes virus gH protein and the recombinant bovine herpes virus gL protein form a heterodimer.

The invention also provides an expressed gene composition, which comprises at least two of gB gene, gD gene, gH gene and gL gene; the gB, gD, gH and gL genes encode the bovine herpes virus recombinant gB, gD, gH and gL proteins, respectively.

In one embodiment, the nucleotide sequences of the gB gene, the gD gene, the gH gene and the gL gene are shown as SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

The invention also provides an expression vector system comprising one or more vectors for expressing the bovine herpes virus antigen composition described above.

In one embodiment, the vector contains the gB gene and the gD gene, and the gB gene and the gD gene are linked by an IRES sequence; or the vector contains the gH gene and the gL gene, and the gH gene and the gL gene are linked by an IRES sequence.

The invention also provides a host cell system comprising one or more cells for expressing the bovine herpes virus antigen composition described above.

The invention also provides application of the bovine herpes virus antigen composition, the expression gene composition, the expression vector system or the host cell system in preparing a product for preventing and treating infectious bovine rhinotracheitis.

The invention also provides a bovine infectious rhinotracheitis vaccine which comprises the bovine herpes virus antigen composition and a pharmaceutically acceptable adjuvant.

In one embodiment, the adjuvant is one or more of MONTANIDE ISA 206 VG, MONTANIDE ISA 201 VG, liquid paraffin, camphor oil, and plant cell agglutinin.

In one embodiment, the adjuvant is montainide ISA 201 VG.

The invention comprehensively analyzes the sequence information and the spatial structure of the gB protein, the gD protein, the gH protein and the gL protein of the bovine herpes virus, and carries out site-directed mutagenesis on the four proteins respectively. After mutation, the respective immunogenicity of the proteins is improved, and stable heterodimer proteins can be formed between the mutated gB protein and the mutated gD protein and between the mutated gH protein and the mutated gL protein, and are similar to dimers formed by the gB protein, the gD protein, the gH protein and the gL protein when natural wild viruses infect cells, and are close to the natural structural state of the proteins. Thus, the neutralizing antibody can be better stimulated, and the immune effect is better and the immune period is longer than that of the method of using the proteins respectively or directly mixing the proteins. The bovine herpes virus antigen composition has antigenicity, immunogenicity and functions similar to those of virus natural proteins, is high in expression level, and has strong immunogenicity, long immune period, no pathogenicity to cattle, high safety, strong humoral immunity in cattle and capacity of resisting strong virus and attacking virus. And the bovine herpes virus antigen composition can be prepared by large-scale serum-free suspension culture in a bioreactor, so that the production cost of the vaccine is greatly reduced.

Drawings

FIG. 1 is a gel electrophoresis chart of the PCR amplification product of the gB-gD gene in example 1, in which the band of interest appeared at the 2.9kbp position;

FIG. 2 is a gel electrophoresis chart of the PCR-amplified product of the colony in example 1, in which the band of interest appears at the position of 2.9 kbp;

FIG. 3 is a schematic diagram showing the structure of the transfer vector pCI-gB-gD-GS containing a target gene in example 1;

FIG. 4 is a gel electrophoresis photograph of the PCR-amplified product of the gH-gL gene of example 2, in which the band of interest appeared at the 2.7kbp position;

FIG. 5 is a gel electrophoresis chart of the PCR-amplified product of the colony in example 2, wherein the band of interest appears at the position of 2.7 kbp;

FIG. 6 is a schematic structural view of the transfer vector pCI-gH-gL-GS containing a target gene in example 2;

FIG. 7 is a non-reducing SDS-PAGE detection profile of the cell culture obtained in example 4;

FIG. 8 is a reducing SDS-PAGE profile of the cell culture obtained in example 4;

FIG. 9 is a Western Blot analysis of the product after non-reducing SDS-PAGE in example 5;

FIG. 10 is a Western Blot analysis of the product after the reducing SDS-PAGE electrophoresis in example 5.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Interpretation of terms

"antigen" refers to all substances that induce the immune response of the body, i.e., substances that are specifically recognized and bound by antigen receptors (TCR/BCR) on the surface of T/B lymphocytes, activate T/B cells, proliferate and differentiate to produce immune response products (sensitized lymphocytes or antibodies), and specifically bind to the corresponding products in vitro and in vivo. Thus, antigens have two basic properties, namely antigenicity and immunogenicity. Antigenicity refers to the ability of an antigen to specifically bind to the antibody or sensitized lymphocyte it induces. Immunogenicity refers to the property of eliciting an immune response, i.e., the ability of an antigen to stimulate a specific immune cell, activate, proliferate, differentiate the immune cell, and ultimately produce immune effector antibodies and sensitized lymphocytes.

"vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40).

"host cell" means a cell which can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

A bovine herpes virus antigen composition of an embodiment of the invention includes at least two of the following proteins: a recombinant bovine herpes virus gB protein with an amino acid sequence shown as SEQ ID NO. 1, a recombinant bovine herpes virus gD protein with an amino acid sequence shown as SEQ ID NO. 2, a recombinant bovine herpes virus gH protein with an amino acid sequence shown as SEQ ID NO. 3 and a recombinant bovine herpes virus gL protein with an amino acid sequence shown as SEQ ID NO. 4.

In one particular example, a bovine herpes virus antigen composition includes a bovine herpes virus recombinant gB protein and a bovine herpes virus recombinant gD protein. In one particular example, a bovine herpes virus antigen composition includes a bovine herpes virus recombinant gH protein and a bovine herpes virus recombinant gL protein.

In one particular example, the bovine herpes virus antigen composition includes a bovine herpes virus recombinant gB protein, a bovine herpes virus recombinant gD protein, a bovine herpes virus recombinant gH protein, and a bovine herpes virus recombinant gL protein. The four proteins are used together, so that a better immune effect can be obtained.

In a specific example, the recombinant bovine herpes virus gB protein and the recombinant bovine herpes virus gD protein in the bovine herpes virus antigen composition form a heterodimer, and the recombinant bovine herpes virus gH protein and the recombinant bovine herpes virus gL protein form a heterodimer. Thus, the heterodimeric protein formed is similar to the dimer formed when the natural wild virus infects cells, is close to the natural structural state of the protein, and can better stimulate neutralizing antibodies.

The expressed gene composition of one embodiment of the present invention includes at least two of the gB gene, the gD gene, the gH gene, and the gL gene. The gB gene, gD gene, gH gene and gL gene encode the recombinant bovine herpes virus gB protein, recombinant bovine herpes virus gD protein, recombinant bovine herpes virus gH protein and recombinant bovine herpes virus gL protein, respectively.

In a specific example, the nucleotide sequences of the gB gene, the gD gene, the gH gene, and the gL gene are shown as SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, and SEQ ID NO 8, respectively. It will be appreciated that due to the degeneracy of the codons, the nucleic acid sequences capable of expressing the same protein have a variety of forms, the above being codon optimized nucleic acid sequences, but are not limited thereto.

The expression vector system of one embodiment of the invention comprises one or more vectors for expressing the bovine herpes virus antigen composition described above. It will be appreciated that one vector may contain one or more of the gB gene, gD gene, gH gene and gL gene, and that the four proteins may be expressed separately or together as desired.

In a specific example, the vector contains a gB gene and a gD gene, and the gB gene and the gD gene are linked by an IRES sequence; alternatively, the vector contains the gH gene and the gL gene, and the gH gene and the gL gene are linked by an IRES sequence. Thus, the recombinant gB protein and the recombinant gD protein, or the recombinant gH protein and the recombinant gL protein can be co-expressed by using one expression cassette, and the heterodimeric protein is directly formed after expression.

It will be appreciated that the vector may also contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.). In a specific example, the initial vector may be selected from pSV2-GS, pCI-GS, pcDNA4-GS, etc., preferably pCI-GS.

The host cell system of one embodiment of the invention comprises one or more cells for expressing the bovine herpes virus antigen composition described above. It is understood that one cell may contain one or more of the above genes or the above vectors.

In one particular example, the host cell is a CHO cell. Specifically, the CHO cell line may be DG44, DXB11, CHO-K1, CHO-S cell line, etc., preferably CHO-S. The CHO cell is used for expressing the modified bovine herpes virus recombinant protein, the glycosylation of eukaryotic expression protein is sufficient, the immunogenicity of antigen protein is good, the expression quantity is very high, the recombinant cell can be cultured in a suspension way on a large scale, the complexity of vaccine preparation is greatly reduced, and the production cost is reduced.

The preparation method of the bovine herpes virus recombinant protein provided by the embodiment of the invention comprises the following steps: culturing the host cell under appropriate conditions, collecting the culture solution and/or the host cell lysate, and then separating and purifying to obtain the recombinant bovine herpes virus protein.

In a specific example, the method of separation and purification includes nickel column affinity chromatography, molecular sieve chromatography, and the like, but is not limited thereto and may be selected as needed.

The infectious bovine rhinotracheitis vaccine provided by the embodiment of the invention comprises the bovine herpes virus antigen composition and a pharmaceutically acceptable adjuvant.

In one specific example, the adjuvant can be MONTANIDE ISA 206 VG, MONTANIDE ISA 201 VG, liquid paraffin, camphor oil, plant cell agglutinin, etc., or a combination of two or more, preferably MONTANIDE ISA 201 VG is used.

Embodiments of the present invention will be described in detail below with reference to specific examples.

Example 1 construction of recombinant eukaryotic expression vector pCI-gB-gD-GS

gB-gD gene expression cassette amplification and purification

A codon-optimized gB-gD gene expression cassette (SEQ ID NO: 9) was synthesized in Nanjing Kingsri Biotechnology Ltd and cloned into a pUC-57 vector to obtain a pUC-gB-gD plasmid vector. PCR amplification was performed using pUC-gB-gD as a template and gB-gD-F, gB-gD-R as a primer, and the amplification system is shown in Table 1. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 95 ℃ for 45 seconds, renaturation at 60 ℃ for 45 seconds, extension at 72 ℃ for 2 minutes, 30 cycles; extension at 72 ℃ for 10 minutes and storage at 4 ℃.

gB-gD-F：5’-ATAGGTACCgccgccaccatggaaaccgataccctgctgctgtgggtgctgctg-3’

gB-gD-R：5’-ATACTCGAGttaatgatgatgatgatgatggccttccgggccGCAcgggccgccgttg-3’

TABLE 1 gB-gD Gene expression cassette amplification System

The PCR product was subjected to gel electrophoresis to identify the size of the target gene, and as shown in FIG. 1, a band appeared at a position of about 2.9kbp, and the target gene was successfully amplified, and then recovered and purified using a gel recovery and purification kit.

2. Enzyme digestion

The PCR products of the pCI-GS plasmid and the purified gB-gD gene expression cassette were digested with Xho I and Kpn I at 37 ℃ for 3 hours, respectively, and the reaction systems are shown in tables 2 and 3. And respectively recovering enzyme digestion products after gel electrophoresis, and purifying by using a gel recovery and purification kit.

TABLE 2 gB-gD Gene expression cassette enzyme digestion reaction System

TABLE 3 pCI-GS plasmid digestion reaction System

3. Connection of

The digested pCI-GS plasmid and gB-gD gene expression cassette were ligated overnight using T4 DNA ligase in a 16 ℃ water bath, the ligation system is shown in Table 4.

TABLE 4 ligation system of gB-gD gene expression cassette and pCI-GS plasmid

4. Transformation of

mu.L of the ligation product was added to 100. mu.L of DH 5. alpha. competent cells, mixed well, heat-shocked at 42 ℃ for 90 seconds, ice-bathed for 2 minutes, added to 900. mu.L of LB medium without Amp, and cultured at 37 ℃ for 1 hour. 1.0ml of the cell suspension was concentrated by centrifugation to 100. mu.L, applied to LB solid medium containing Amp, and cultured at 37 ℃ for 16 hours.

5. Colony PCR and sequencing identification

And (3) selecting single colonies on the plate, respectively inoculating the single colonies into an LB liquid culture medium, culturing for 2 hours at 37 ℃, and carrying out colony PCR by using a bacterial liquid as a template and gB-gD-F and gB-gD-R as primers. The size of the gene of interest was confirmed by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 2, a sample showing a band of approximately 2.9kbp was positive. And (4) sending the bacterial liquid with positive colony PCR identification to a sequencing company for sequencing, and selecting the bacterial liquid with correct sequencing for storage. Obtaining the eukaryotic expression vector pCI-gB-gD-GS. The map of the constructed vector is shown in FIG. 3.

Example 2 construction of recombinant eukaryotic expression vector pCI-gH-gL-GS

Amplification and purification of gH-gL gene expression cassette

The codon-optimized gH-gL gene expression cassette (SEQ ID NO: 10) was synthesized by Shanghai Sangni Biotechnology Co., Ltd and cloned into a pUC-57 vector to obtain a pUC-gH-gL plasmid vector. PCR amplification was performed using pUC-gH-gL as a template and gH-gL-F, gH-gL-R as a primer, and the amplification system is shown in Table 5. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 95 ℃ for 45 seconds, renaturation at 60 ℃ for 45 seconds, extension at 72 ℃ for 2 minutes, 30 cycles; extension at 72 ℃ for 10 minutes and storage at 4 ℃.

gH-gL-F：5’-ATAGGTACCGCCGCCACCatggaaaccgataccctgctgctgtgggtgctgctg-3’

gH-gL-R：5’-ATACTCGAGttaatgatgatgatgatgatggcgataaatgccatcgc-3’

TABLE 5 gH-gL Gene expression cassette amplification System

The PCR product was subjected to gel electrophoresis to identify the size of the target gene, and as shown in FIG. 4, a band appeared at a position of about 2.7kbp, and the target gene was successfully amplified, and then recovered and purified using a gel recovery and purification kit.

2. Enzyme digestion

The PCR products of the pCI-GS plasmid and the purified gH-gL gene expression cassette were digested with Xho I and Kpn I at 37 ℃ for 3 hours, respectively, and the reaction systems are shown in tables 6 and 7. And respectively recovering enzyme digestion products after gel electrophoresis, and purifying by using a gel recovery and purification kit.

TABLE 6 gH-gL Gene expression cassette enzyme digestion reaction System

TABLE 7 pCI-GS plasmid digestion reaction System

3. Connection of

The digested pCI-GS plasmid and the digested gH-gL gene expression cassette were ligated with T4 DNA ligase in a 16 ℃ water bath overnight, and the ligation system is shown in Table 8.

TABLE 8 ligation system of gH-gL gene expression cassette and pCI-GS plasmid

4. Transformation of

5. Colony PCR and sequencing identification

And (3) selecting single colonies on the plate, respectively inoculating the single colonies into an LB liquid culture medium, culturing for 2 hours at 37 ℃, and carrying out colony PCR by using a bacterial liquid as a template and gH-gL-F and gH-gL-R as primers. The size of the gene of interest was confirmed by subjecting the PCR product to gel electrophoresis, and as shown in FIG. 5, a sample showing a band of approximately 2.7kbp was positive. And (4) sending the bacterial liquid with positive colony PCR identification to a sequencing company for sequencing, and selecting the bacterial liquid with correct sequencing for storage. Obtaining the eukaryotic expression vector pCI-gH-gL-GS. The map of the constructed vector is shown in FIG. 6.

Example 3 construction and screening of recombinant CHO cells

1. Cell transfection

1.1 preparation of cells CHO cells in logarithmic growth phase were sampled and counted at 1X 10⁶continuously passaging the cells/mL, maintaining the seeds, centrifuging the rest cells, centrifuging at 1000rpm for 4 minutes, discarding the supernatant, re-suspending the supernatant by using about 20mL of fresh CHO-WM medium, centrifuging again, centrifuging at 1000rpm for 4 minutes, discarding the supernatant, re-suspending and counting the supernatant by using a small amount of medium, and finally adjusting the cell density to 1.43 multiplied by 10⁷cells/mL。

1.2 mixing of plasmid and cells 5. mu.g of the pCI-gB-gD-GS plasmid vector of example 1 was taken, and added to an EP tube, 0.7mL of cells was added, and after mixing uniformly, the mixture was left to stand for 15 minutes.

1.3 electric shock 2 pulses of 280V 20ms, immediately transferring the cells into a shake flask after the electric shock is finished, performing suspension culture, observing the cell state after 48h, changing the culture solution, and growing the cells to 0.6 × 10 when the cell density is up to 0.6 × 10⁶For cells/mL, 50. mu.M MSX (L-methionine sulphoxide) was added for pressure screening.

2. Monoclonal screening

2.1 resuspend cells in CHO cell serum-free protein free media CHO-WM cell media + 50. mu.M MSX from Volmer Biotechnology Ltd, Suzhou, and count.

2.2 plating to dilute the cells to 5/mL, add 200. mu.L of the mixed cells to a 96-well plate, stand at 37 ℃ with 5% CO₂And incubating for 4-6 h in the cell incubator. Wells of individual cells were recorded.

2.3 when the wells of a single cell in the 96-well plate were grown up, the medium was discarded, PBS was washed once, 100. mu.L of 0.25% trypsin-EDTA was digested at room temperature for about 2min, 2mL of CHO-WM medium (containing 10% FBS + 50. mu.M MSX) was added to stop the digestion reaction, and the cells were blown off with a pipette. And transferring the cells to a 12-pore plate, taking the supernatant when the 12-pore plate is full, detecting whether the clone is positive by Elisa, continuously carrying out expanded culture on the high-efficiency expression positive clone, and freezing and storing.

3. Cell shake flask fermentation

3.1 subculture medium configuration: CHO-WM medium was used to add 50. mu.M MSX as subculture medium and placed in a 37 ℃ water bath to preheat to 37 ℃.

3.2 from CO₂Taking out the shake flask cells by a constant temperature shaking table, and counting.

3.3 dilution of the cells to (2.5-3.5). times.10⁵cells/mL were inoculated in 30mL culture medium in a 125mL shake flask. The cell culture flask was placed at 37 ℃ with 5% CO₂Incubate overnight in a constant temperature shaker at 100 rpm/min.

3.4 counting the cell density and the cell activity every 24 hours, measuring the glucose, and adding the glucose to 4g/L when the sugar is lower than 2 g/L; samples were taken at 1mL per day and the supernatant was used to detect protein expression.

Recombinant CHO cells co-expressing the gH-gL protein were constructed and selected in the same manner.

Cell lines expressing the proteins shown in tables 9 and 10 were also constructed according to the above example method.

TABLE 9

Watch 10

Example 4 SDS-PAGE detection

The cell culture supernatants of the gB-gD, gH-gL and gH-gL groups harvested in example 3 were subjected to non-reducing SDS-PAGE, while the cell culture supernatants of the gB-gD and gH-gL groups were subjected to reducing SDS-PAGE, and empty CHO cells were used as a negative control (reducing vs. non-reducing ones were distinguished by whether or not the reducing agent β -mercaptoethanol was added at the time of sample treatment). The specific operation is as follows: mu.L of the harvested cell culture was taken, 10. mu.L of 5 Xloading buffer was added to 1.25mL of 1mol/L Tris-HCI (pH6.8), 25mg of bromophenol blue, 2.5mL of glycerol, 0.5g of SDS in ddH₂And O, diluting to 5mL, subpackaging with 0.5 mL/tube, storing at room temperature, adding 25 mu L of beta-mercaptoethanol into each tube before using for reducibility detection, uniformly mixing, carrying out boiling water bath for 5 minutes, centrifuging at 12000r/min for 1 minute, taking supernatant, carrying out SDS-PAGE gel (12% concentration gel) electrophoresis, taking gel after electrophoresis, dyeing and decoloring, and observing target strips.

As shown in FIGS. 7 and 8, in the non-reducing SDS-PAGE assay, the gB-gD group and the gB-gD group showed bands around molecular weights of about 105kDa, 60kDa and 40kDa, the gH-gL group and the gH-gL group showed bands around molecular weights of about 90kDa, 60kDa and 22kDa, the band of the dimer formed between the two proteins was significant, the gB-gD group showed more dimers than the gB-gD group, the gH-gL group showed more dimers than the gH-gL group, and the negative control showed no band at the corresponding position. In the reduced SDS-PAGE detection, the gB-gD group showed only bands around the molecular weights of about 60kDa and 40kDa, the gH-gL group showed only bands around the molecular weights of about 60kDa and 22kDa, and the negative control showed no band at the corresponding position.

Example 5 Western Blot assay

Products obtained after SDS-PAGE in example 4 are respectively transferred to an NC (nitrocellulose) membrane, and are sealed by 5% skimmed milk for 2 hours, and the bovine-derived anti-IBRV positive serum is incubated for 2 hours, rinsed, and a goat anti-bovine polyclonal antibody marked by HRP is incubated for 2 hours, rinsed, and then added with an enhanced chemiluminescence fluorescent substrate dropwise, and a photograph is taken by using a chemiluminescence imager. The results are shown in FIGS. 9 and 10, in which the recombinant CHO supernatant sample has a target band, and the negative control has no target band, indicating that the target antigen protein is correctly expressed in the recombinant CHO cells.

Example 6 protein content and agar detection

The gB-gD and gH-gL protein content in the CHO cell culture supernatants harvested in example 3 were determined using the Elisa method. The operation mode is as follows: bovine anti-IBRV multi-antiserum was diluted with coating buffer to appropriate concentration, 100 μ L per well, overnight at 4 ℃, washed three times with PBST, and blocked with 1% BSA for 1 h. Adding antigen standard substances (protein obtained by ion exchange chromatography, hydrophobic chromatography and molecular sieve purification) with different concentrations and diluting the sample to be detected in a gradient manner, incubating for 1 hour at 37 ℃, and washing with PBST for three times. Monoclonal antibodies for detecting gB-gD and gH-gL proteins were added to each well, incubated at 37 ℃ for 1 hour, and washed three times with PBST. A secondary antibody, HRP-labeled goat anti-bovine IgG, was added to each well, incubated at 37 ℃ for 1 hour, and washed three times with PBST. TMB development for 10 min, 2M H₂SO₄The reaction was terminated. Reading by a microplate reader, and calculating the amount of gB-gD and gH-gL proteins in the sample to be detected through a standard curve.

The average contents of gB-gD protein and gH-gL protein in the vaccine stock solution reached 2.4g/L and 2.3g/L, respectively, as determined by Elisa of large-scale preparation of gB-gD and gH-gL proteins according to example 3.

Detecting the titer of expressed gB-gD and gH-gL proteins by using an agar expansion method, drilling a plum blossom hole on an agarose gel plate, adding IBRV agar expansion detection standard serum in the middle of the plum blossom hole, and adding 2-diluted expression antigens of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 power around the plum blossom hole respectively. And (5) observing a precipitation line after inverted incubation for 72h, wherein the maximum dilution ratio of the precipitation line is the agar-agar titer. The agar titer detection results are as follows: the agar titer of the gB-gD protein and the agar titer of the gH-gL protein are respectively 1:512 and 1: 256.

Example 7 vaccine preparation

The cell lines prepared in example 3 were grouped and prepared into vaccines as shown in table 11, and the specific operations were as follows: and mixing the proteins expressed by the appropriate amount of CHO cells in each group in equal proportion, adding the mixture into MONTANIDE ISA 201 VG adjuvant (volume ratio is 46: 54), so that the concentration of the proteins in the finally emulsified vaccine is 100 mug/mL, and storing at 4 ℃ after emulsification and quality inspection are qualified.

TABLE 11

Example 8 Final vaccine testing

Test one: safety inspection

The animal experiment substitute selects 6 healthy rabbits of 1.5-2.0 kg, wherein 4 rabbits in an immunization group, 2 rabbits in a blank group, 1.0mL of vaccine in the immunization group injected into the leg muscle of the rabbits in the immunization group, and 1.0mL of adjuvant injected into the leg muscle of the rabbits in the blank group, the body temperature is measured continuously for 7 days and at a fixed point every afternoon, no death and adverse reaction occur, no clinical abnormal expression occurs, and the body temperature is normal. The method comprises the steps of selecting 6 healthy female guinea pigs of 350-400 g, wherein 4 immune groups and 2 control groups are selected, 0.5mL of immune group vaccine is injected subcutaneously into each neck of the immune group guinea pigs, 0.5mL of adjuvant is injected subcutaneously into each neck of the blank group guinea pigs, and the continuous observation for 7 days has no death and adverse reaction and no clinical abnormal expression.

And (2) test II: efficacy test

(1) Elisa antibody detection: screening 55 heads of 4-5-month-old calves (IBRV antigen-antibody negative), randomly dividing into 11 groups, injecting 2mL of vaccine into muscles according to the table 11 for 5 heads of each group, boosting immunity once after three weeks of priming immunization, collecting serum before immunization, before secondary immunization and 21 days after secondary immunization, and detecting the titer of the antibody. The results are shown in Table 12, and the results using the bovine viral diarrhea virus antibody detection kit of IDEXX company show that: the modified bovine herpes virus recombinant protein can obtain better immune effect, and the effect is still remarkable after 21 days of secondary immunization.

TABLE 12

And (4) judging a result: criteria for judging antibody positivity: P/N is more than or equal to 2.1, OD450 is more than or equal to 0.1

(2) And (3) immunizing and attacking poison of cattle: the test selects 30 healthy susceptible cattle with 2-3 months old IBRV antigen and antibody double negative (the antibody negative is that the serum neutralizing antibody titer is less than or equal to 1:4 or the ELISA detects the antibody negative), 10 cattle are respectively selected from an immune group, a control group 2 and a blank group, three groups of cattle are respectively injected with 2.0mL through neck muscles according to the corresponding group of a table 11, the immunity is enhanced in the same way after 21 days, IBRV virulent challenge is carried out 21 days after the secondary immunization, 2.5mL IBRV virulent strains are respectively dripped into the nostrils in the morning and afternoon of each cattle, 14 days are continuously observed after the virulent challenge, the body temperature is measured at fixed points every morning, the clinical symptoms are observed, and the nasal swabs are collected for pathogen detection and virus separation. The immune group had 9 cattle protection, the control group 2 had 5 cattle protection, and the blank group had 9 cattle.

TABLE 13 IBRV potency assay (bovine immune challenge) results

Note: the disease is judged to be the disease according to two items of the rising body temperature, the nasal discharge and the toxic brought by the nasal swab.

The relevant sequence information is as follows:

recombinant gB protein (SEQ ID NO: 1):

METDTLLLWVLLLWVPGSTGGMGEITDLANKKWRCLSKAEYLRSGRKVVAFDRDDDPWEAPLKPARLSAPGVRGWHTTDDVYTALGSAGLYRTGTSVNCIVEEVEARSVYPYDSFAFSTGDIIYMSPFYGLREGAHREHTSYSPERFQQIEGYYKRDMATGRRLKEPVSRNFLRTQHVTVAWDWVPKRKNVCSLAKWREADEMLRDESRGNFRFTARSLSATFVSDSHTFALQNVPLSDCVIEEAEAAVERVYRERYNGTHVLSGSLETYLARGGFVVAFRPMLSNELAKLYLQELARSNGTLEGLFAAAAPKPGPRRARRAAPSAPGGPGAANGPAGDGDAGGRVTTVSSAEFAALQFTYDHIQDHVNTMFSRLATSWCLLQNKERALWAEAAKLNPSAAASAALDRRAAARMLGCAMAVTYCHELGEGRVFIENSMRAPGGVCYSRPPVSFAFGNESECVEGQLGEDNELLPGRELVEPCTAN

recombinant gD protein (SEQ ID NO: 2):

METDTLLLWVLLLWVPGSTGHTTGPIPSPFADGREQPVEVRYATSAAACDMLALIADPQVGRTLWEAVRRHARAYNATVIWYKIESGCARPLYYMEYTECEPRKHFGYCRYRTPPFWDSFLAGFAYPTDDELGLIMAAPARLVEGQYRRALYIDGTVAYTDFMVSLPAGDCWFSKLGAARGYTFGACFPARDYEQKKVLRLTYLTQYYPQEAHKAIVDYWFMRHGGVVPSYFEESKGYEPPPAADGGSCAPPGDDEAREDEGETEDGAAGREGNGGPCGPEG

recombinant gH protein (SEQ ID NO: 3):

METDTLLLWVLLLWVPGSTGFSQARAESNAARPPPAPRVTPTPAGRVAAFDINDVLASGPEHFFVPVRADRKRRERHVADFAAVWPVSYIPAGRAVLSCERAAARLAVGLGFLSVSVTSRDLLPLEFMVAPADANVRMITAFNGGGAFPPPGPAAGPQRRAYVIGYGNSRLDSHMYLTMREVASYANEPADFRAHLTAAHREAFLMLREAAAARRGPSAGPAPNAAYHAYRVAARLGLALSALTEGALADGYVLAEELVDLDYHLKLLSRVLLGAGLGCAANGRVRARTIAQLAVPRELRPDAFIPEPAGAALESVVARGRKLRAVYAFSGPDAPLAARLLAHGVVSDLYDAFLRGELTWGPPMRHALFFAVAASAFPADAQALELARDVTRKCTAMCTAGHATAAALDLEEVYAHVGGGAGGDAGFELLDAFSPCMASFRLDLLEEAHVLDVLSAVPARAALDAWLECQPAAAAPNLSAAALGMLGRGGLFGPAHAAALAPELFAAPCGGWGAGAAVAIVPVAPNASYVITRAHPRRGLTY

recombinant gL protein (SEQ ID NO: 4):

METDTLLLWVLLLWVPGSTGTRRADSAESILAERCRGNLLLADRPQHEEAAPGLAGIFIRGRCSPPEAALWYEDTGETYWANPYAVARGLAEDIRRVLADTPVYRDLAIQVLNSAFGLPHEVRAPLPPPPRGCVLPPCYHTTGPCGPGDGIYR

recombinant gB protein gene (SEQ ID NO: 5):

atggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggcggcatgggcgaaattaccgatctggcgaacaaaaaatggcgctgcctgagcaaagcggaatatctgcgcagcggccgcaaagtggtggcgtttgatcgcgatgatgatccgtgggaagcgccgctgaaaccggcgcgcctgagcgcgccgggcgtgcgcggctggcataccaccgatgatgtgtataccgcgctgggcagcgcgggcctgtatcgcaccggcaccagcgtgaactgcattgtggaagaagtggaagcgcgcagcgtgtatccgtatgatagctttgcgtttagcaccggcgatattatttatatgagcccgttttatggcctgcgcgaaggcgcgcatcgcgaacataccagctatagcccggaacgctttcagcagattgaaggctattataaacgcgatatggcgaccggccgccgcctgaaagaaccggtgagccgcaactttctgcgcacccagcatgtgaccgtggcgtgggattgggtgccgaaacgcaaaaacgtgtgcagcctggcgaaatggcgcgaagcggatgaaatgctgcgcgatgaaagccgcggcaactttcgctttaccgcgcgcagcctgagcgcgacctttgtgagcgatagccatacctttgcgctgcagaacgtgccgctgagcgattgcgtgattgaagaagcggaagcggcggtggaacgcgtgtatcgcgaacgctataacggcacccatgtgctgagcggcagcctggaaacctatctggcgcgcggcggctttgtggtggcgtttcgcccgatgctgagcaacgaactggcgaaactgtatctgcaggaactggcgcgcagcaacggcaccctggaaggcctgtttgcggcggcggcgccgaaaccgggcccgcgccgcgcgcgccgcgcggcgccgagcgcgccgggcggcccgggcgcggcgaacggcccggcgggcgatggcgatgcgggcggccgcgtgaccaccgtgagcagcgcggaatttgcggcgctgcagtttacctatgatcatattcaggatcatgtgaacaccatgtttagccgcctggcgaccagctggtgcctgctgcagaacaaagaacgcgcgctgtgggcggaagcggcgaaactgaacccgagcgcggcggcgagcgcggcgctggatcgccgcgcggcggcgcgcatgctgggcTGCgcgatggcggtgacctattgccatgaactgggcgaaggccgcgtgtttattgaaaacagcatgcgcgcgccgggcggcgtgtgctatagccgcccgccggtgagctttgcgtttggcaacgaaagcgaaTGCgtggaaggccagctgggcgaagataacgaactgctgccgggccgcgaactggtggaaccgtgcaccgcgaac

recombinant gD protein gene (SEQ ID NO: 6):

atggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggccataccaccggcccgattccgagcccgtttgcggatggccgcgaacagccggtggaagtgcgctatgcgaccagcgcggcggcgtgcgatatgctggcgctgattgcggatccgcaggtgggccgcaccctgtgggaagcggtgcgccgccatgcgcgcgcgtataacgcgaccgtgatttggtataaaattgaaagcggctgcgcgcgcccgctgtattatatggaatataccgaatgcgaaccgcgcaaacattttggctattgccgctatcgcaccccgccgttttgggatagctttctggcgggctttgcgtatccgaccgatgatgaactgggcctgattatggcggcgccggcgcgcctggtggaaggccagtatcgccgcgcgctgtatattgatggcaccgtggcgtataccgattttatggtgagcctgccggcgggcgattgctggtttagcaaactgggcgcggcgcgcggctatacctttggcgcgtgctttccggcgcgcgattatgaacagaaaaaagtgctgcgcctgacctatctgacccagtattatccgcaggaagcgcataaagcgattgtggattattggtttatgcgccatggcggcgtggtgccgagctattttgaagaaagcaaaggctatgaaccgccgccggcggcggatggcggcagcTGCgcgccgccgggcgatgatgaagcgcgcgaagatgaaggcgaaaccgaagatggcgcggcgggccgcgaaggcaacggcggcccgTGCggcccggaaggc

recombinant gH protein gene (SEQ ID NO: 7):

atggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggctttagccaggcgcgcgcggaaagcaacgcggcgcgcccgccgccggcgccgcgcgtgaccccgaccccggcgggccgcgtggcggcgtttgatattaacgatgtgctggcgagcggcccggaacatttttttgtgccggtgcgcgcggatcgcaaacgccgcgaacgccatgtggcggattttgcggcggtgtggccggtgagctatattccggcgggccgcgcggtgctgagctgcgaacgcgcggcggcgcgcctggcggtgggcctgggctttctgagcgtgagcgtgaccagccgcgatctgctgccgctggaatttatggtggcgccggcggatgcgaacgtgcgcatgattaccgcgtttaacggcggcggcgcgtttccgccgccgggcccggcggcgggcccgcagcgccgcgcgtatgtgattggctatggcaacagccgcctggatagccatatgtatctgaccatgcgcgaagtggcgagctatgcgaacgaaccggcggattttcgcgcgcatctgaccgcggcgcatcgcgaagcgtttctgatgctgcgcgaagcggcggcggcgcgccgcggcccgagcgcgggcccggcgccgaacgcggcgtatcatgcgtatcgcgtggcggcgcgcctgggcctggcgctgagcgcgctgaccgaaggcgcgctggcggatggctatgtgctggcggaagaactggtggatctggattatcatctgaaactgctgagccgcgtgctgctgggcgcgggcctgggctgcgcggcgaacggccgcgtgcgcgcgcgcaccattgcgcagctggcggtgccgcgcgaactgcgcccggatgcgtttattccggaaccggcgggcgcggcgctggaaagcgtggtggcgcgcggccgcaaactgcgcgcggtgtatgcgtttagcggcccggatgcgccgctggcggcgcgcctgctggcgcatggcgtggtgagcgatctgtatgatgcgtttctgcgcggcgaactgacctggggcccgccgatgcgccatgcgctgttttttgcggtggcggcgagcgcgtttccggcggatgcgcaggcgctggaactggcgcgcgatgtgacccgcaaatgcaccgcgatgtgcaccgcgggccatgcgaccgcggcggcgctggatctggaagaagtgtatgcgcatgtgggcggcggcgcgggcggcgatgcgggctttgaactgctggatgcgtttagcccgtgcatggcgagctttcgcctggatctgctggaagaagcgcatgtgctggatgtgctgagcgcggtgccggcgcgcgcggcgctggatgcgtggctggaaTGTcagccggcggcggcggcgccgaacctgagcgcggcggcgctgggcatgctgggccgcggcggcctgtttggcccggcgcatgcggcggcgctggcgccggaactgtttgcggcgccgtgcggcggctggggcgcgggcgcggcggtggcgattgtgccggtggcgccgaacgcgagctatgtgattacccgcgcgcatccgcgccgcggcctgacctat

recombinant gL protein gene (SEQ ID NO: 8):

atggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggcacccgccgcgcggatagcgcggaaagcattctggcggaacgctgccgcggcaacctgctgctggcggatcgcccgcagcatgaagaagcggcgccgggcctggcgggcatttttattcgcggccgctgcagcccgccggaagcggcgctgtggtatgaagataccggcgaaacctattgggcgaacccgtatgcggtggcgcgcggcctggcggaagatattcgccgcgtgctggcggataccccggtgtatcgcgatctggcgattcaggtgctgaacagcgcgtttggcctgccgcatgaagtgcgcgcgccgctgccgccgccgccgcgcggctgcgtgctgccgccgTGTtatcataccaccggcccgtgcggcccgggcgatggcatttatcgc

IRES sequence:

cgcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa

gB-gD gene expression cassette (SEQ ID NO: 9):

gccgccaccatggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggcggcatgggcgaaattaccgatctggcgaacaaaaaatggcgctgcctgagcaaagcggaatatctgcgcagcggccgcaaagtggtggcgtttgatcgcgatgatgatccgtgggaagcgccgctgaaaccggcgcgcctgagcgcgccgggcgtgcgcggctggcataccaccgatgatgtgtataccgcgctgggcagcgcgggcctgtatcgcaccggcaccagcgtgaactgcattgtggaagaagtggaagcgcgcagcgtgtatccgtatgatagctttgcgtttagcaccggcgatattatttatatgagcccgttttatggcctgcgcgaaggcgcgcatcgcgaacataccagctatagcccggaacgctttcagcagattgaaggctattataaacgcgatatggcgaccggccgccgcctgaaagaaccggtgagccgcaactttctgcgcacccagcatgtgaccgtggcgtgggattgggtgccgaaacgcaaaaacgtgtgcagcctggcgaaatggcgcgaagcggatgaaatgctgcgcgatgaaagccgcggcaactttcgctttaccgcgcgcagcctgagcgcgacctttgtgagcgatagccatacctttgcgctgcagaacgtgccgctgagcgattgcgtgattgaagaagcggaagcggcggtggaacgcgtgtatcgcgaacgctataacggcacccatgtgctgagcggcagcctggaaacctatctggcgcgcggcggctttgtggtggcgtttcgcccgatgctgagcaacgaactggcgaaactgtatctgcaggaactggcgcgcagcaacggcaccctggaaggcctgtttgcggcggcggcgccgaaaccgggcccgcgccgcgcgcgccgcgcggcgccgagcgcgccgggcggcccgggcgcggcgaacggcccggcgggcgatggcgatgcgggcggccgcgtgaccaccgtgagcagcgcggaatttgcggcgctgcagtttacctatgatcatattcaggatcatgtgaacaccatgtttagccgcctggcgaccagctggtgcctgctgcagaacaaagaacgcgcgctgtgggcggaagcggcgaaactgaacccgagcgcggcggcgagcgcggcgctggatcgccgcgcggcggcgcgcatgctgggcTGCgcgatggcggtgacctattgccatgaactgggcgaaggccgcgtgtttattgaaaacagcatgcgcgcgccgggcggcgtgtgctatagccgcccgccggtgagctttgcgtttggcaacgaaagcgaaTGCgtggaaggccagctgggcgaagataacgaactgctgccgggccgcgaactggtggaaccgtgcaccgcgaaccatcatcatcatcatcattaacgcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttccttt gaaaaacacgatgataagccgccaccatggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggccataccaccggcccgattccgagcccgtttgcggatggccgcgaacagccggtggaagtgcgctatgcgaccagcgcggcggcgtgcgatatgctggcgctgattgcggatccgcaggtgggccgcaccctgtgggaagcggtgcgccgccatgcgcgcgcgtataacgcgaccgtgatttggtataaaattgaaagcggctgcgcgcgcccgctgtattatatggaatataccgaatgcgaaccgcgcaaacattttggctattgccgctatcgcaccccgccgttttgggatagctttctggcgggctttgcgtatccgaccgatgatgaactgggcctgattatggcggcgccggcgcgcctggtggaaggccagtatcgccgcgcgctgtatattgatggcaccgtggcgtataccgattttatggtgagcctgccggcgggcgattgctggtttagcaaactgggcgcggcgcgcggctatacctttggcgcgtgctttccggcgcgcgattatgaacagaaaaaagtgctgcgcctgacctatctgacccagtattatccgcaggaagcgcataaagcgattgtggattattggtttatgcgccatggcggcgtggtgccgagctattttgaagaaagcaaaggctatgaaccgccgccggcggcggatggcggcagcTGCgcgccgccgggcgatgatgaagcgcgcgaagatgaaggcgaaaccgaagatggcgcggcgggccgcgaaggcaacggcggcccgTGCggcccggaaggccatcatcatcatcatcattaa

gH-gL gene expression cassette (SEQ ID NO: 10):

gccgccaccatggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggctttagccaggcgcgcgcggaaagcaacgcggcgcgcccgccgccggcgccgcgcgtgaccccgaccccggcgggccgcgtggcggcgtttgatattaacgatgtgctggcgagcggcccggaacatttttttgtgccggtgcgcgcggatcgcaaacgccgcgaacgccatgtggcggattttgcggcggtgtggccggtgagctatattccggcgggccgcgcggtgctgagctgcgaacgcgcggcggcgcgcctggcggtgggcctgggctttctgagcgtgagcgtgaccagccgcgatctgctgccgctggaatttatggtggcgccggcggatgcgaacgtgcgcatgattaccgcgtttaacggcggcggcgcgtttccgccgccgggcccggcggcgggcccgcagcgccgcgcgtatgtgattggctatggcaacagccgcctggatagccatatgtatctgaccatgcgcgaagtggcgagctatgcgaacgaaccggcggattttcgcgcgcatctgaccgcggcgcatcgcgaagcgtttctgatgctgcgcgaagcggcggcggcgcgccgcggcccgagcgcgggcccggcgccgaacgcggcgtatcatgcgtatcgcgtggcggcgcgcctgggcctggcgctgagcgcgctgaccgaaggcgcgctggcggatggctatgtgctggcggaagaactggtggatctggattatcatctgaaactgctgagccgcgtgctgctgggcgcgggcctgggctgcgcggcgaacggccgcgtgcgcgcgcgcaccattgcgcagctggcggtgccgcgcgaactgcgcccggatgcgtttattccggaaccggcgggcgcggcgctggaaagcgtggtggcgcgcggccgcaaactgcgcgcggtgtatgcgtttagcggcccggatgcgccgctggcggcgcgcctgctggcgcatggcgtggtgagcgatctgtatgatgcgtttctgcgcggcgaactgacctggggcccgccgatgcgccatgcgctgttttttgcggtggcggcgagcgcgtttccggcggatgcgcaggcgctggaactggcgcgcgatgtgacccgcaaatgcaccgcgatgtgcaccgcgggccatgcgaccgcggcggcgctggatctggaagaagtgtatgcgcatgtgggcggcggcgcgggcggcgatgcgggctttgaactgctggatgcgtttagcccgtgcatggcgagctttcgcctggatctgctggaagaagcgcatgtgctggatgtgctgagcgcggtgccggcgcgcgcggcgctggatgcgtggctggaaTGTcagccggcggcggcggcgccgaacctgagcgcggcggcgctgggcatgctgggccgcggcggcctgtttggcccggcgcatgcggcggcgctggcgccggaactgtttgcggcgccgtgcggcggctggggcgcgggcgcggcggtggcgattgtgccggtggcgccgaacgcgagctatgtgattacccgcgcgcatccgcgccgcggcctgacctatcatcatcatcatcatcattgacgcccctc tccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataagccgccaccatggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggcacccgccgcgcggatagcgcggaaagcattctggcggaacgctgccgcggcaacctgctgctggcggatcgcccgcagcatgaagaagcggcgccgggcctggcgggcatttttattcgcggccgctgcagcccgccggaagcggcgctgtggtatgaagataccggcgaaacctattgggcgaacccgtatgcggtggcgcgcggcctggcggaagatattcgccgcgtgctggcggataccccggtgtatcgcgatctggcgattcaggtgctgaacagcgcgtttggcctgccgcatgaagtgcgcgcgccgctgccgccgccgccgcgcggctgcgtgctgccgccgTGTtatcataccaccggcccgtgcggcccgggcgatggcatttatcgccatcatcatcatcatcattaa

unmutated gB protein gene:

atggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggcggcatgggcgaaattaccgatctggcgaacaaaaaatggcgctgcctgagcaaagcggaatatctgcgcagcggccgcaaagtggtggcgtttgatcgcgatgatgatccgtgggaagcgccgctgaaaccggcgcgcctgagcgcgccgggcgtgcgcggctggcataccaccgatgatgtgtataccgcgctgggcagcgcgggcctgtatcgcaccggcaccagcgtgaactgcattgtggaagaagtggaagcgcgcagcgtgtatccgtatgatagctttgcgtttagcaccggcgatattatttatatgagcccgttttatggcctgcgcgaaggcgcgcatcgcgaacataccagctatagcccggaacgctttcagcagattgaaggctattataaacgcgatatggcgaccggccgccgcctgaaagaaccggtgagccgcaactttctgcgcacccagcatgtgaccgtggcgtgggattgggtgccgaaacgcaaaaacgtgtgcagcctggcgaaatggcgcgaagcggatgaaatgctgcgcgatgaaagccgcggcaactttcgctttaccgcgcgcagcctgagcgcgacctttgtgagcgatagccatacctttgcgctgcagaacgtgccgctgagcgattgcgtgattgaagaagcggaagcggcggtggaacgcgtgtatcgcgaacgctataacggcacccatgtgctgagcggcagcctggaaacctatctggcgcgcggcggctttgtggtggcgtttcgcccgatgctgagcaacgaactggcgaaactgtatctgcaggaactggcgcgcagcaacggcaccctggaaggcctgtttgcggcggcggcgccgaaaccgggcccgcgccgcgcgcgccgcgcggcgccgagcgcgccgggcggcccgggcgcggcgaacggcccggcgggcgatggcgatgcgggcggccgcgtgaccaccgtgagcagcgcggaatttgcggcgctgcagtttacctatgatcatattcaggatcatgtgaacaccatgtttagccgcctggcgaccagctggtgcctgctgcagaacaaagaacgcgcgctgtgggcggaagcggcgaaactgaacccgagcgcggcggcgagcgcggcgctggatcgccgcgcggcggcgcgcatgctgggcGATgcgatggcggtgacctattgccatgaactgggcgaaggccgcgtgtttattgaaaacagcatgcgcgcgccgggcggcgtgtgctatagccgcccgccggtgagctttgcgtttggcaacgaaagcgaaCCGgtggaaggccagctgggcgaagataacgaactgctgccgggccgcgaactggtggaaccgtgcaccgcgaac

unmutated gD protein gene:

atggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggccataccaccggcccgattccgagcccgtttgcggatggccgcgaacagccggtggaagtgcgctatgcgaccagcgcggcggcgtgcgatatgctggcgctgattgcggatccgcaggtgggccgcaccctgtgggaagcggtgcgccgccatgcgcgcgcgtataacgcgaccgtgatttggtataaaattgaaagcggctgcgcgcgcccgctgtattatatggaatataccgaatgcgaaccgcgcaaacattttggctattgccgctatcgcaccccgccgttttgggatagctttctggcgggctttgcgtatccgaccgatgatgaactgggcctgattatggcggcgccggcgcgcctggtggaaggccagtatcgccgcgcgctgtatattgatggcaccgtggcgtataccgattttatggtgagcctgccggcgggcgattgctggtttagcaaactgggcgcggcgcgcggctatacctttggcgcgtgctttccggcgcgcgattatgaacagaaaaaagtgctgcgcctgacctatctgacccagtattatccgcaggaagcgcataaagcgattgtggattattggtttatgcgccatggcggcgtggtgccgagctattttgaagaaagcaaaggctatgaaccgccgccggcggcggatggcggcagcCCGgcgccgccgggcgatgatgaagcgcgcgaagatgaaggcgaaaccgaagatggcgcggcgggccgcgaaggcaacggcggcccgCCGggcccggaaggc

unmutated gH protein gene:

atggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggctttagccaggcgcgcgcggaaagcaacgcggcgcgcccgccgccggcgccgcgcgtgaccccgaccccggcgggccgcgtggcggcgtttgatattaacgatgtgctggcgagcggcccggaacatttttttgtgccggtgcgcgcggatcgcaaacgccgcgaacgccatgtggcggattttgcggcggtgtggccggtgagctatattccggcgggccgcgcggtgctgagctgcgaacgcgcggcggcgcgcctggcggtgggcctgggctttctgagcgtgagcgtgaccagccgcgatctgctgccgctggaatttatggtggcgccggcggatgcgaacgtgcgcatgattaccgcgtttaacggcggcggcgcgtttccgccgccgggcccggcggcgggcccgcagcgccgcgcgtatgtgattggctatggcaacagccgcctggatagccatatgtatctgaccatgcgcgaagtggcgagctatgcgaacgaaccggcggattttcgcgcgcatctgaccgcggcgcatcgcgaagcgtttctgatgctgcgcgaagcggcggcggcgcgccgcggcccgagcgcgggcccggcgccgaacgcggcgtatcatgcgtatcgcgtggcggcgcgcctgggcctggcgctgagcgcgctgaccgaaggcgcgctggcggatggctatgtgctggcggaagaactggtggatctggattatcatctgaaactgctgagccgcgtgctgctgggcgcgggcctgggctgcgcggcgaacggccgcgtgcgcgcgcgcaccattgcgcagctggcggtgccgcgcgaactgcgcccggatgcgtttattccggaaccggcgggcgcggcgctggaaagcgtggtggcgcgcggccgcaaactgcgcgcggtgtatgcgtttagcggcccggatgcgccgctggcggcgcgcctgctggcgcatggcgtggtgagcgatctgtatgatgcgtttctgcgcggcgaactgacctggggcccgccgatgcgccatgcgctgttttttgcggtggcggcgagcgcgtttccggcggatgcgcaggcgctggaactggcgcgcgatgtgacccgcaaatgcaccgcgatgtgcaccgcgggccatgcgaccgcggcggcgctggatctggaagaagtgtatgcgcatgtgggcggcggcgcgggcggcgatgcgggctttgaactgctggatgcgtttagcccgtgcatggcgagctttcgcctggatctgctggaagaagcgcatgtgctggatgtgctgagcgcggtgccggcgcgcgcggcgctggatgcgtggctggaaGCGcagccggcggcggcggcgccgaacctgagcgcggcggcgctgggcatgctgggccgcggcggcctgtttggcccggcgcatgcggcggcgctggcgccggaactgtttgcggcgccgtgcggcggctggggcgcgggcgcggcggtggcgattgtgccggtggcgccgaacgcgagctatgtgattacccgcgcgcatccgcgccgcggcctgacctat

unmutated gL protein gene:

atggaaaccgataccctgctgctgtgggtgctgctgctgtgggtgccgggcagcaccggcacccgccgcgcggatagcgcggaaagcattctggcggaacgctgccgcggcaacctgctgctggcggatcgcccgcagcatgaagaagcggcgccgggcctggcgggcatttttattcgcggccgctgcagcccgccggaagcggcgctgtggtatgaagataccggcgaaacctattgggcgaacccgtatgcggtggcgcgcggcctggcggaagatattcgccgcgtgctggcggataccccggtgtatcgcgatctggcgattcaggtgctgaacagcgcgtttggcctgccgcatgaagtgcgcgcgccgctgccgccgccgccgcgcggctgcgtgctgccgccgCGCtatcataccaccggcccgtgcggcccgggcgatggcatttatcgc

the technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Suzhou Shino Biotechnology Ltd

<120> bovine herpes virus antigen composition and use thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 485

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Gly Met Gly Glu Ile Thr Asp Leu Ala Asn Lys Lys

20 25 30

Trp Arg Cys Leu Ser Lys Ala Glu Tyr Leu Arg Ser Gly Arg Lys Val

35 40 45

Val Ala Phe Asp Arg Asp Asp Asp Pro Trp Glu Ala Pro Leu Lys Pro

50 55 60

Ala Arg Leu Ser Ala Pro Gly Val Arg Gly Trp His Thr Thr Asp Asp

65 70 75 80

Val Tyr Thr Ala Leu Gly Ser Ala Gly Leu Tyr Arg Thr Gly Thr Ser

85 90 95

Val Asn Cys Ile Val Glu Glu Val Glu Ala Arg Ser Val Tyr Pro Tyr

100 105 110

Asp Ser Phe Ala Phe Ser Thr Gly Asp Ile Ile Tyr Met Ser Pro Phe

115 120 125

Tyr Gly Leu Arg Glu Gly Ala His Arg Glu His Thr Ser Tyr Ser Pro

130 135 140

Glu Arg Phe Gln Gln Ile Glu Gly Tyr Tyr Lys Arg Asp Met Ala Thr

145 150 155 160

Gly Arg Arg Leu Lys Glu Pro Val Ser Arg Asn Phe Leu Arg Thr Gln

165 170 175

His Val Thr Val Ala Trp Asp Trp Val Pro Lys Arg Lys Asn Val Cys

180 185 190

Ser Leu Ala Lys Trp Arg Glu Ala Asp Glu Met Leu Arg Asp Glu Ser

195 200 205

Arg Gly Asn Phe Arg Phe Thr Ala Arg Ser Leu Ser Ala Thr Phe Val

210 215 220

Ser Asp Ser His Thr Phe Ala Leu Gln Asn Val Pro Leu Ser Asp Cys

225 230 235 240

Val Ile Glu Glu Ala Glu Ala Ala Val Glu Arg Val Tyr Arg Glu Arg

245 250 255

Tyr Asn Gly Thr His Val Leu Ser Gly Ser Leu Glu Thr Tyr Leu Ala

260 265 270

Arg Gly Gly Phe Val Val Ala Phe Arg Pro Met Leu Ser Asn Glu Leu

275 280 285

Ala Lys Leu Tyr Leu Gln Glu Leu Ala Arg Ser Asn Gly Thr Leu Glu

290 295 300

Gly Leu Phe Ala Ala Ala Ala Pro Lys Pro Gly Pro Arg Arg Ala Arg

305 310 315 320

Arg Ala Ala Pro Ser Ala Pro Gly Gly Pro Gly Ala Ala Asn Gly Pro

325 330 335

Ala Gly Asp Gly Asp Ala Gly Gly Arg Val Thr Thr Val Ser Ser Ala

340 345 350

Glu Phe Ala Ala Leu Gln Phe Thr Tyr Asp His Ile Gln Asp His Val

355 360 365

Asn Thr Met Phe Ser Arg Leu Ala Thr Ser Trp Cys Leu Leu Gln Asn

370 375 380

Lys Glu Arg Ala Leu Trp Ala Glu Ala Ala Lys Leu Asn Pro Ser Ala

385 390 395 400

Ala Ala Ser Ala Ala Leu Asp Arg Arg Ala Ala Ala Arg Met Leu Gly

405 410 415

Cys Ala Met Ala Val Thr Tyr Cys His Glu Leu Gly Glu Gly Arg Val

420 425 430

Phe Ile Glu Asn Ser Met Arg Ala Pro Gly Gly Val Cys Tyr Ser Arg

435 440 445

Pro Pro Val Ser Phe Ala Phe Gly Asn Glu Ser Glu Cys Val Glu Gly

450 455 460

Gln Leu Gly Glu Asp Asn Glu Leu Leu Pro Gly Arg Glu Leu Val Glu

465 470 475 480

Pro Cys Thr Ala Asn

485

<210> 2

<211> 282

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly His Thr Thr Gly Pro Ile Pro Ser Pro Phe Ala Asp

20 25 30

Gly Arg Glu Gln Pro Val Glu Val Arg Tyr Ala Thr Ser Ala Ala Ala

35 40 45

Cys Asp Met Leu Ala Leu Ile Ala Asp Pro Gln Val Gly Arg Thr Leu

50 55 60

Trp Glu Ala Val Arg Arg His Ala Arg Ala Tyr Asn Ala Thr Val Ile

65 70 75 80

Trp Tyr Lys Ile Glu Ser Gly Cys Ala Arg Pro Leu Tyr Tyr Met Glu

85 90 95

Tyr Thr Glu Cys Glu Pro Arg Lys His Phe Gly Tyr Cys Arg Tyr Arg

100 105 110

Thr Pro Pro Phe Trp Asp Ser Phe Leu Ala Gly Phe Ala Tyr Pro Thr

115 120 125

Asp Asp Glu Leu Gly Leu Ile Met Ala Ala Pro Ala Arg Leu Val Glu

130 135 140

Gly Gln Tyr Arg Arg Ala Leu Tyr Ile Asp Gly Thr Val Ala Tyr Thr

145 150 155 160

Asp Phe Met Val Ser Leu Pro Ala Gly Asp Cys Trp Phe Ser Lys Leu

165 170 175

Gly Ala Ala Arg Gly Tyr Thr Phe Gly Ala Cys Phe Pro Ala Arg Asp

180 185 190

Tyr Glu Gln Lys Lys Val Leu Arg Leu Thr Tyr Leu Thr Gln Tyr Tyr

195 200 205

Pro Gln Glu Ala His Lys Ala Ile Val Asp Tyr Trp Phe Met Arg His

210 215 220

Gly Gly Val Val Pro Ser Tyr Phe Glu Glu Ser Lys Gly Tyr Glu Pro

225 230 235 240

Pro Pro Ala Ala Asp Gly Gly Ser Cys Ala Pro Pro Gly Asp Asp Glu

245 250 255

Ala Arg Glu Asp Glu Gly Glu Thr Glu Asp Gly Ala Ala Gly Arg Glu

260 265 270

Gly Asn Gly Gly Pro Cys Gly Pro Glu Gly

275 280

<210> 3

<211> 542

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Phe Ser Gln Ala Arg Ala Glu Ser Asn Ala Ala Arg

20 25 30

Pro Pro Pro Ala Pro Arg Val Thr Pro Thr Pro Ala Gly Arg Val Ala

35 40 45

Ala Phe Asp Ile Asn Asp Val Leu Ala Ser Gly Pro Glu His Phe Phe

50 55 60

Val Pro Val Arg Ala Asp Arg Lys Arg Arg Glu Arg His Val Ala Asp

65 70 75 80

Phe Ala Ala Val Trp Pro Val Ser Tyr Ile Pro Ala Gly Arg Ala Val

85 90 95

Leu Ser Cys Glu Arg Ala Ala Ala Arg Leu Ala Val Gly Leu Gly Phe

100 105 110

Leu Ser Val Ser Val Thr Ser Arg Asp Leu Leu Pro Leu Glu Phe Met

115 120 125

Val Ala Pro Ala Asp Ala Asn Val Arg Met Ile Thr Ala Phe Asn Gly

130 135 140

Gly Gly Ala Phe Pro Pro Pro Gly Pro Ala Ala Gly Pro Gln Arg Arg

145 150 155 160

Ala Tyr Val Ile Gly Tyr Gly Asn Ser Arg Leu Asp Ser His Met Tyr

165 170 175

Leu Thr Met Arg Glu Val Ala Ser Tyr Ala Asn Glu Pro Ala Asp Phe

180 185 190

Arg Ala His Leu Thr Ala Ala His Arg Glu Ala Phe Leu Met Leu Arg

195 200 205

Glu Ala Ala Ala Ala Arg Arg Gly Pro Ser Ala Gly Pro Ala Pro Asn

210 215 220

Ala Ala Tyr His Ala Tyr Arg Val Ala Ala Arg Leu Gly Leu Ala Leu

225 230 235 240

Ser Ala Leu Thr Glu Gly Ala Leu Ala Asp Gly Tyr Val Leu Ala Glu

245 250 255

Glu Leu Val Asp Leu Asp Tyr His Leu Lys Leu Leu Ser Arg Val Leu

260 265 270

Leu Gly Ala Gly Leu Gly Cys Ala Ala Asn Gly Arg Val Arg Ala Arg

275 280 285

Thr Ile Ala Gln Leu Ala Val Pro Arg Glu Leu Arg Pro Asp Ala Phe

290 295 300

Ile Pro Glu Pro Ala Gly Ala Ala Leu Glu Ser Val Val Ala Arg Gly

305 310 315 320

Arg Lys Leu Arg Ala Val Tyr Ala Phe Ser Gly Pro Asp Ala Pro Leu

325 330 335

Ala Ala Arg Leu Leu Ala His Gly Val Val Ser Asp Leu Tyr Asp Ala

340 345 350

Phe Leu Arg Gly Glu Leu Thr Trp Gly Pro Pro Met Arg His Ala Leu

355 360 365

Phe Phe Ala Val Ala Ala Ser Ala Phe Pro Ala Asp Ala Gln Ala Leu

370 375 380

Glu Leu Ala Arg Asp Val Thr Arg Lys Cys Thr Ala Met Cys Thr Ala

385 390 395 400

Gly His Ala Thr Ala Ala Ala Leu Asp Leu Glu Glu Val Tyr Ala His

405 410 415

Val Gly Gly Gly Ala Gly Gly Asp Ala Gly Phe Glu Leu Leu Asp Ala

420 425 430

Phe Ser Pro Cys Met Ala Ser Phe Arg Leu Asp Leu Leu Glu Glu Ala

435 440 445

His Val Leu Asp Val Leu Ser Ala Val Pro Ala Arg Ala Ala Leu Asp

450 455 460

Ala Trp Leu Glu Cys Gln Pro Ala Ala Ala Ala Pro Asn Leu Ser Ala

465 470 475 480

Ala Ala Leu Gly Met Leu Gly Arg Gly Gly Leu Phe Gly Pro Ala His

485 490 495

Ala Ala Ala Leu Ala Pro Glu Leu Phe Ala Ala Pro Cys Gly Gly Trp

500 505 510

Gly Ala Gly Ala Ala Val Ala Ile Val Pro Val Ala Pro Asn Ala Ser

515 520 525

Tyr Val Ile Thr Arg Ala His Pro Arg Arg Gly Leu Thr Tyr

530 535 540

<210> 4

<211> 153

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Thr Arg Arg Ala Asp Ser Ala Glu Ser Ile Leu Ala

20 25 30

Glu Arg Cys Arg Gly Asn Leu Leu Leu Ala Asp Arg Pro Gln His Glu

35 40 45

Glu Ala Ala Pro Gly Leu Ala Gly Ile Phe Ile Arg Gly Arg Cys Ser

50 55 60

Pro Pro Glu Ala Ala Leu Trp Tyr Glu Asp Thr Gly Glu Thr Tyr Trp

65 70 75 80

Ala Asn Pro Tyr Ala Val Ala Arg Gly Leu Ala Glu Asp Ile Arg Arg

85 90 95

Val Leu Ala Asp Thr Pro Val Tyr Arg Asp Leu Ala Ile Gln Val Leu

100 105 110

Asn Ser Ala Phe Gly Leu Pro His Glu Val Arg Ala Pro Leu Pro Pro

115 120 125

Pro Pro Arg Gly Cys Val Leu Pro Pro Cys Tyr His Thr Thr Gly Pro

130 135 140

Cys Gly Pro Gly Asp Gly Ile Tyr Arg

145 150

<210> 5

<211> 1455

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggaaaccg ataccctgct gctgtgggtg ctgctgctgt gggtgccggg cagcaccggc 60

ggcatgggcg aaattaccga tctggcgaac aaaaaatggc gctgcctgag caaagcggaa 120

tatctgcgca gcggccgcaa agtggtggcg tttgatcgcg atgatgatcc gtgggaagcg 180

ccgctgaaac cggcgcgcct gagcgcgccg ggcgtgcgcg gctggcatac caccgatgat 240

gtgtataccg cgctgggcag cgcgggcctg tatcgcaccg gcaccagcgt gaactgcatt 300

gtggaagaag tggaagcgcg cagcgtgtat ccgtatgata gctttgcgtt tagcaccggc 360

gatattattt atatgagccc gttttatggc ctgcgcgaag gcgcgcatcg cgaacatacc 420

agctatagcc cggaacgctt tcagcagatt gaaggctatt ataaacgcga tatggcgacc 480

ggccgccgcc tgaaagaacc ggtgagccgc aactttctgc gcacccagca tgtgaccgtg 540

gcgtgggatt gggtgccgaa acgcaaaaac gtgtgcagcc tggcgaaatg gcgcgaagcg 600

gatgaaatgc tgcgcgatga aagccgcggc aactttcgct ttaccgcgcg cagcctgagc 660

gcgacctttg tgagcgatag ccataccttt gcgctgcaga acgtgccgct gagcgattgc 720

gtgattgaag aagcggaagc ggcggtggaa cgcgtgtatc gcgaacgcta taacggcacc 780

catgtgctga gcggcagcct ggaaacctat ctggcgcgcg gcggctttgt ggtggcgttt 840

cgcccgatgc tgagcaacga actggcgaaa ctgtatctgc aggaactggc gcgcagcaac 900

ggcaccctgg aaggcctgtt tgcggcggcg gcgccgaaac cgggcccgcg ccgcgcgcgc 960

cgcgcggcgc cgagcgcgcc gggcggcccg ggcgcggcga acggcccggc gggcgatggc 1020

gatgcgggcg gccgcgtgac caccgtgagc agcgcggaat ttgcggcgct gcagtttacc 1080

tatgatcata ttcaggatca tgtgaacacc atgtttagcc gcctggcgac cagctggtgc 1140

ctgctgcaga acaaagaacg cgcgctgtgg gcggaagcgg cgaaactgaa cccgagcgcg 1200

gcggcgagcg cggcgctgga tcgccgcgcg gcggcgcgca tgctgggctg cgcgatggcg 1260

gtgacctatt gccatgaact gggcgaaggc cgcgtgttta ttgaaaacag catgcgcgcg 1320

ccgggcggcg tgtgctatag ccgcccgccg gtgagctttg cgtttggcaa cgaaagcgaa 1380

tgcgtggaag gccagctggg cgaagataac gaactgctgc cgggccgcga actggtggaa 1440

ccgtgcaccg cgaac 1455

<210> 6

<211> 846

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggaaaccg ataccctgct gctgtgggtg ctgctgctgt gggtgccggg cagcaccggc 60

cataccaccg gcccgattcc gagcccgttt gcggatggcc gcgaacagcc ggtggaagtg 120

cgctatgcga ccagcgcggc ggcgtgcgat atgctggcgc tgattgcgga tccgcaggtg 180

ggccgcaccc tgtgggaagc ggtgcgccgc catgcgcgcg cgtataacgc gaccgtgatt 240

tggtataaaa ttgaaagcgg ctgcgcgcgc ccgctgtatt atatggaata taccgaatgc 300

gaaccgcgca aacattttgg ctattgccgc tatcgcaccc cgccgttttg ggatagcttt 360

ctggcgggct ttgcgtatcc gaccgatgat gaactgggcc tgattatggc ggcgccggcg 420

cgcctggtgg aaggccagta tcgccgcgcg ctgtatattg atggcaccgt ggcgtatacc 480

gattttatgg tgagcctgcc ggcgggcgat tgctggttta gcaaactggg cgcggcgcgc 540

ggctatacct ttggcgcgtg ctttccggcg cgcgattatg aacagaaaaa agtgctgcgc 600

ctgacctatc tgacccagta ttatccgcag gaagcgcata aagcgattgt ggattattgg 660

tttatgcgcc atggcggcgt ggtgccgagc tattttgaag aaagcaaagg ctatgaaccg 720

ccgccggcgg cggatggcgg cagctgcgcg ccgccgggcg atgatgaagc gcgcgaagat 780

gaaggcgaaa ccgaagatgg cgcggcgggc cgcgaaggca acggcggccc gtgcggcccg 840

gaaggc 846

<210> 7

<211> 1626

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggaaaccg ataccctgct gctgtgggtg ctgctgctgt gggtgccggg cagcaccggc 60

tttagccagg cgcgcgcgga aagcaacgcg gcgcgcccgc cgccggcgcc gcgcgtgacc 120

ccgaccccgg cgggccgcgt ggcggcgttt gatattaacg atgtgctggc gagcggcccg 180

gaacattttt ttgtgccggt gcgcgcggat cgcaaacgcc gcgaacgcca tgtggcggat 240

tttgcggcgg tgtggccggt gagctatatt ccggcgggcc gcgcggtgct gagctgcgaa 300

cgcgcggcgg cgcgcctggc ggtgggcctg ggctttctga gcgtgagcgt gaccagccgc 360

gatctgctgc cgctggaatt tatggtggcg ccggcggatg cgaacgtgcg catgattacc 420

gcgtttaacg gcggcggcgc gtttccgccg ccgggcccgg cggcgggccc gcagcgccgc 480

gcgtatgtga ttggctatgg caacagccgc ctggatagcc atatgtatct gaccatgcgc 540

gaagtggcga gctatgcgaa cgaaccggcg gattttcgcg cgcatctgac cgcggcgcat 600

cgcgaagcgt ttctgatgct gcgcgaagcg gcggcggcgc gccgcggccc gagcgcgggc 660

ccggcgccga acgcggcgta tcatgcgtat cgcgtggcgg cgcgcctggg cctggcgctg 720

agcgcgctga ccgaaggcgc gctggcggat ggctatgtgc tggcggaaga actggtggat 780

ctggattatc atctgaaact gctgagccgc gtgctgctgg gcgcgggcct gggctgcgcg 840

gcgaacggcc gcgtgcgcgc gcgcaccatt gcgcagctgg cggtgccgcg cgaactgcgc 900

ccggatgcgt ttattccgga accggcgggc gcggcgctgg aaagcgtggt ggcgcgcggc 960

cgcaaactgc gcgcggtgta tgcgtttagc ggcccggatg cgccgctggc ggcgcgcctg 1020

ctggcgcatg gcgtggtgag cgatctgtat gatgcgtttc tgcgcggcga actgacctgg 1080

ggcccgccga tgcgccatgc gctgtttttt gcggtggcgg cgagcgcgtt tccggcggat 1140

gcgcaggcgc tggaactggc gcgcgatgtg acccgcaaat gcaccgcgat gtgcaccgcg 1200

ggccatgcga ccgcggcggc gctggatctg gaagaagtgt atgcgcatgt gggcggcggc 1260

gcgggcggcg atgcgggctt tgaactgctg gatgcgttta gcccgtgcat ggcgagcttt 1320

cgcctggatc tgctggaaga agcgcatgtg ctggatgtgc tgagcgcggt gccggcgcgc 1380

gcggcgctgg atgcgtggct ggaatgtcag ccggcggcgg cggcgccgaa cctgagcgcg 1440

gcggcgctgg gcatgctggg ccgcggcggc ctgtttggcc cggcgcatgc ggcggcgctg 1500

gcgccggaac tgtttgcggc gccgtgcggc ggctggggcg cgggcgcggc ggtggcgatt 1560

gtgccggtgg cgccgaacgc gagctatgtg attacccgcg cgcatccgcg ccgcggcctg 1620

acctat 1626

<210> 8

<211> 459

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggaaaccg ataccctgct gctgtgggtg ctgctgctgt gggtgccggg cagcaccggc 60

acccgccgcg cggatagcgc ggaaagcatt ctggcggaac gctgccgcgg caacctgctg 120

ctggcggatc gcccgcagca tgaagaagcg gcgccgggcc tggcgggcat ttttattcgc 180

ggccgctgca gcccgccgga agcggcgctg tggtatgaag ataccggcga aacctattgg 240

gcgaacccgt atgcggtggc gcgcggcctg gcggaagata ttcgccgcgt gctggcggat 300

accccggtgt atcgcgatct ggcgattcag gtgctgaaca gcgcgtttgg cctgccgcat 360

gaagtgcgcg cgccgctgcc gccgccgccg cgcggctgcg tgctgccgcc gtgttatcat 420

accaccggcc cgtgcggccc gggcgatggc atttatcgc 459

<210> 9

<211> 2937

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gccgccacca tggaaaccga taccctgctg ctgtgggtgc tgctgctgtg ggtgccgggc 60

agcaccggcg gcatgggcga aattaccgat ctggcgaaca aaaaatggcg ctgcctgagc 120

aaagcggaat atctgcgcag cggccgcaaa gtggtggcgt ttgatcgcga tgatgatccg 180

tgggaagcgc cgctgaaacc ggcgcgcctg agcgcgccgg gcgtgcgcgg ctggcatacc 240

accgatgatg tgtataccgc gctgggcagc gcgggcctgt atcgcaccgg caccagcgtg 300

aactgcattg tggaagaagt ggaagcgcgc agcgtgtatc cgtatgatag ctttgcgttt 360

agcaccggcg atattattta tatgagcccg ttttatggcc tgcgcgaagg cgcgcatcgc 420

gaacatacca gctatagccc ggaacgcttt cagcagattg aaggctatta taaacgcgat 480

atggcgaccg gccgccgcct gaaagaaccg gtgagccgca actttctgcg cacccagcat 540

gtgaccgtgg cgtgggattg ggtgccgaaa cgcaaaaacg tgtgcagcct ggcgaaatgg 600

cgcgaagcgg atgaaatgct gcgcgatgaa agccgcggca actttcgctt taccgcgcgc 660

agcctgagcg cgacctttgt gagcgatagc catacctttg cgctgcagaa cgtgccgctg 720

agcgattgcg tgattgaaga agcggaagcg gcggtggaac gcgtgtatcg cgaacgctat 780

aacggcaccc atgtgctgag cggcagcctg gaaacctatc tggcgcgcgg cggctttgtg 840

gtggcgtttc gcccgatgct gagcaacgaa ctggcgaaac tgtatctgca ggaactggcg 900

cgcagcaacg gcaccctgga aggcctgttt gcggcggcgg cgccgaaacc gggcccgcgc 960

cgcgcgcgcc gcgcggcgcc gagcgcgccg ggcggcccgg gcgcggcgaa cggcccggcg 1020

ggcgatggcg atgcgggcgg ccgcgtgacc accgtgagca gcgcggaatt tgcggcgctg 1080

cagtttacct atgatcatat tcaggatcat gtgaacacca tgtttagccg cctggcgacc 1140

agctggtgcc tgctgcagaa caaagaacgc gcgctgtggg cggaagcggc gaaactgaac 1200

ccgagcgcgg cggcgagcgc ggcgctggat cgccgcgcgg cggcgcgcat gctgggctgc 1260

gcgatggcgg tgacctattg ccatgaactg ggcgaaggcc gcgtgtttat tgaaaacagc 1320

atgcgcgcgc cgggcggcgt gtgctatagc cgcccgccgg tgagctttgc gtttggcaac 1380

gaaagcgaat gcgtggaagg ccagctgggc gaagataacg aactgctgcc gggccgcgaa 1440

ctggtggaac cgtgcaccgc gaaccatcat catcatcatc attaacgccc ctctccctcc 1500

ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat 1560

atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct 1620

gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg 1680

ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta 1740

gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag 1800

ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg 1860

atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat 1920

gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca 1980

tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc 2040

ctttgaaaaa cacgatgata agccgccacc atggaaaccg ataccctgct gctgtgggtg 2100

ctgctgctgt gggtgccggg cagcaccggc cataccaccg gcccgattcc gagcccgttt 2160

gcggatggcc gcgaacagcc ggtggaagtg cgctatgcga ccagcgcggc ggcgtgcgat 2220

atgctggcgc tgattgcgga tccgcaggtg ggccgcaccc tgtgggaagc ggtgcgccgc 2280

catgcgcgcg cgtataacgc gaccgtgatt tggtataaaa ttgaaagcgg ctgcgcgcgc 2340

ccgctgtatt atatggaata taccgaatgc gaaccgcgca aacattttgg ctattgccgc 2400

tatcgcaccc cgccgttttg ggatagcttt ctggcgggct ttgcgtatcc gaccgatgat 2460

gaactgggcc tgattatggc ggcgccggcg cgcctggtgg aaggccagta tcgccgcgcg 2520

ctgtatattg atggcaccgt ggcgtatacc gattttatgg tgagcctgcc ggcgggcgat 2580

tgctggttta gcaaactggg cgcggcgcgc ggctatacct ttggcgcgtg ctttccggcg 2640

cgcgattatg aacagaaaaa agtgctgcgc ctgacctatc tgacccagta ttatccgcag 2700

gaagcgcata aagcgattgt ggattattgg tttatgcgcc atggcggcgt ggtgccgagc 2760

tattttgaag aaagcaaagg ctatgaaccg ccgccggcgg cggatggcgg cagctgcgcg 2820

ccgccgggcg atgatgaagc gcgcgaagat gaaggcgaaa ccgaagatgg cgcggcgggc 2880

cgcgaaggca acggcggccc gtgcggcccg gaaggccatc atcatcatca tcattaa 2937

<210> 10

<211> 2721

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gccgccacca tggaaaccga taccctgctg ctgtgggtgc tgctgctgtg ggtgccgggc 60

agcaccggct ttagccaggc gcgcgcggaa agcaacgcgg cgcgcccgcc gccggcgccg 120

cgcgtgaccc cgaccccggc gggccgcgtg gcggcgtttg atattaacga tgtgctggcg 180

agcggcccgg aacatttttt tgtgccggtg cgcgcggatc gcaaacgccg cgaacgccat 240

gtggcggatt ttgcggcggt gtggccggtg agctatattc cggcgggccg cgcggtgctg 300

agctgcgaac gcgcggcggc gcgcctggcg gtgggcctgg gctttctgag cgtgagcgtg 360

accagccgcg atctgctgcc gctggaattt atggtggcgc cggcggatgc gaacgtgcgc 420

atgattaccg cgtttaacgg cggcggcgcg tttccgccgc cgggcccggc ggcgggcccg 480

cagcgccgcg cgtatgtgat tggctatggc aacagccgcc tggatagcca tatgtatctg 540

accatgcgcg aagtggcgag ctatgcgaac gaaccggcgg attttcgcgc gcatctgacc 600

gcggcgcatc gcgaagcgtt tctgatgctg cgcgaagcgg cggcggcgcg ccgcggcccg 660

agcgcgggcc cggcgccgaa cgcggcgtat catgcgtatc gcgtggcggc gcgcctgggc 720

ctggcgctga gcgcgctgac cgaaggcgcg ctggcggatg gctatgtgct ggcggaagaa 780

ctggtggatc tggattatca tctgaaactg ctgagccgcg tgctgctggg cgcgggcctg 840

ggctgcgcgg cgaacggccg cgtgcgcgcg cgcaccattg cgcagctggc ggtgccgcgc 900

gaactgcgcc cggatgcgtt tattccggaa ccggcgggcg cggcgctgga aagcgtggtg 960

gcgcgcggcc gcaaactgcg cgcggtgtat gcgtttagcg gcccggatgc gccgctggcg 1020

gcgcgcctgc tggcgcatgg cgtggtgagc gatctgtatg atgcgtttct gcgcggcgaa 1080

ctgacctggg gcccgccgat gcgccatgcg ctgttttttg cggtggcggc gagcgcgttt 1140

ccggcggatg cgcaggcgct ggaactggcg cgcgatgtga cccgcaaatg caccgcgatg 1200

tgcaccgcgg gccatgcgac cgcggcggcg ctggatctgg aagaagtgta tgcgcatgtg 1260

ggcggcggcg cgggcggcga tgcgggcttt gaactgctgg atgcgtttag cccgtgcatg 1320

gcgagctttc gcctggatct gctggaagaa gcgcatgtgc tggatgtgct gagcgcggtg 1380

ccggcgcgcg cggcgctgga tgcgtggctg gaatgtcagc cggcggcggc ggcgccgaac 1440

ctgagcgcgg cggcgctggg catgctgggc cgcggcggcc tgtttggccc ggcgcatgcg 1500

gcggcgctgg cgccggaact gtttgcggcg ccgtgcggcg gctggggcgc gggcgcggcg 1560

gtggcgattg tgccggtggc gccgaacgcg agctatgtga ttacccgcgc gcatccgcgc 1620

cgcggcctga cctatcatca tcatcatcat cattgacgcc cctctccctc ccccccccct 1680

aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt 1740

tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg 1800

acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc 1860

gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt 1920

tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta 1980

taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg 2040

gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag 2100

gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag 2160

tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa 2220

acacgatgat aagccgccac catggaaacc gataccctgc tgctgtgggt gctgctgctg 2280

tgggtgccgg gcagcaccgg cacccgccgc gcggatagcg cggaaagcat tctggcggaa 2340

cgctgccgcg gcaacctgct gctggcggat cgcccgcagc atgaagaagc ggcgccgggc 2400

ctggcgggca tttttattcg cggccgctgc agcccgccgg aagcggcgct gtggtatgaa 2460

gataccggcg aaacctattg ggcgaacccg tatgcggtgg cgcgcggcct ggcggaagat 2520

attcgccgcg tgctggcgga taccccggtg tatcgcgatc tggcgattca ggtgctgaac 2580

agcgcgtttg gcctgccgca tgaagtgcgc gcgccgctgc cgccgccgcc gcgcggctgc 2640

gtgctgccgc cgtgttatca taccaccggc ccgtgcggcc cgggcgatgg catttatcgc 2700

catcatcatc atcatcatta a 2721

Claims

1. A bovine herpesvirus antigen composition comprising the following proteins: a recombinant bovine herpes virus gB protein with an amino acid sequence shown as SEQ ID NO. 1, a recombinant bovine herpes virus gD protein with an amino acid sequence shown as SEQ ID NO. 2, a recombinant bovine herpes virus gH protein with an amino acid sequence shown as SEQ ID NO. 3 and a recombinant bovine herpes virus gL protein with an amino acid sequence shown as SEQ ID NO. 4.

2. The bovine herpes virus antigen composition of claim 1, wherein the bovine herpes virus recombinant gB protein and the bovine herpes virus recombinant gD protein form a heterodimer and the bovine herpes virus recombinant gH protein and the bovine herpes virus recombinant gL protein form a heterodimer.

3. An expressed gene composition comprising a gB gene, a gD gene, a gH gene, and a gL gene; the gB gene, gD gene, gH gene, and gL gene encode the recombinant bovine herpes virus gB protein, the recombinant bovine herpes virus gD protein, the recombinant bovine herpes virus gH protein, and the recombinant bovine herpes virus gL protein of claim 1, respectively.

4. The expressed gene composition of claim 3, wherein the nucleotide sequences of the gB gene, the gD gene, the gH gene and the gL gene are shown as SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

5. An expression vector system comprising one or more vectors for expressing the bovine herpes virus antigen composition of any one of claims 1-2.

6. The expression vector system according to claim 5, wherein the vector contains the gB gene and the gD gene of claim 3, and the gB gene and the gD gene are linked by an IRES sequence; or the vector contains the gH gene and the gL gene in claim 3, and the gH gene and the gL gene are linked by an IRES sequence.

7. A host cell system comprising one or more cells capable of expressing the bovine herpes virus antigen composition of any one of claims 1-2.

8. Use of a bovine herpes virus antigen composition according to any one of claims 1 to 2, an expressed gene composition according to claim 3 or 4, an expression vector system according to claim 5 or 6 or a host cell system according to claim 7 for the manufacture of a product for the prevention or treatment of infectious bovine rhinotracheitis.

9. A bovine infectious rhinotracheitis vaccine comprising the bovine herpes virus antigen composition of any one of claims 1-2 and a pharmaceutically acceptable adjuvant.

10. The infectious bovine rhinotracheitis vaccine of claim 9, wherein said adjuvant is one or more of MONTANIDE ISA 206 VG, MONTANIDE ISA 201 VG, liquid paraffin, camphor oil, and plant cell agglutinin.

11. The infectious bovine rhinotracheitis vaccine of claim 9, wherein said adjuvant is montainide ISA 201 VG.