CN109134669B - Fusion protein of porcine pseudorabies virus, preparation method, application and vaccine thereof - Google Patents

Abstract

The invention relates to the technical field of biology, and particularly provides a fusion protein of porcine pseudorabies virus, and a preparation method, application and vaccine thereof. The fusion protein of the porcine pseudorabies virus provided by the invention comprises a gB segment and a gD segment, wherein the gB segment is expressed by a nucleotide sequence shown in SEQ ID NO.1, and the gD segment is expressed by a nucleotide sequence shown in SEQ ID NO. 2. The sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are sequences obtained by selecting the genes of a classical strain and a current epidemic strain as research objects and performing comparative analysis, and the sequences are optimized and modified by codons, so that the aims of further improving the broad-spectrum property of the fusion protein antigen and improving the antigen expression quantity are fulfilled. The invention also provides a preparation method and application of the fusion protein and a prepared vaccine.

Description

Fusion protein of porcine pseudorabies virus, preparation method, application and vaccine thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to a fusion protein of porcine pseudorabies virus, and a preparation method, application and vaccine thereof.

Background

Porcine Pseudorabies (PR) is one of important infectious diseases caused by Pseudorabies virus (PRV) and seriously harming the healthy development of the pig industry in China. The virus belongs to a linear double-stranded DNA virus of a herpesviridae subfamily A, and the G + C content of virus DNA is relatively high. At present, 11 types of viral glycoproteins, i.e., essential glycoproteins and non-essential glycoproteins, are known.

Pseudorabies has a wide susceptibility range, and newborn piglets are mostly infected lethally, similar to other susceptible species and mostly die from central nervous system diseases. Adult pig infected with the swine infectious disease mostly shows respiratory diseases, mostly shows recessive infection and has no obvious symptoms. After PRV infection, the immune system of the pig is damaged, the immunity is reduced, and other diseases such as swine fever, porcine reproductive and respiratory syndrome and the like are more easily developed. Increasing the difficulty for preventing and treating the porcine pseudorabies. At present, the infection and incidence rate of PR are high, and huge economic loss is easily caused.

In the case of suckling piglets within 2 weeks of age, symptoms such as fever, anorexia, vomiting, diarrhea, lassitude and the like may appear at the initial stage of infection; with the deepening of the disease, symptoms of neurologic clinical diagnosis such as trembling, ataxia and falling down and limb movement appear; often accompanied by epileptiform seizures or muscle twitching, lethargy, and finally exhaustion death; the mortality rate can reach 100 percent.

The major clinical symptoms of the slightly-large pigs aged about 3-4 weeks are the same as those of piglets aged 2 weeks, but the disease course of the slightly-large pigs is slightly longer than that of piglets aged 2 weeks, the constipation phenomenon is accompanied, and the death rate can generally reach 40-60%; even if the disease is endured, the disease is accompanied by sequelae such as growth retardation, obstruction, hemiplegia and the like.

The immunity of the pigs of more than 2 months old is enhanced, the pigs are mostly subjected to recessive infection after being infected with PRV, and no obvious clinical symptoms exist; the symptoms are mild, and the symptoms of transient fever and cough can also be vomiting.

The pregnant sow has high PRV susceptibility, and symptoms of respiratory system and digestive system such as cough, mental depression, anorexia and the like are shown after infection, and then abortion symptoms, mummy fetus production, stillbirth fetus production and weak fetus production are shown, wherein the stillbirth fetus is taken as the main factor. The weak piglets are often accompanied by symptoms of diarrhea, spasm, dyskinesia, opisthotonus and the like, and die within 24-36 h.

The prevention and control of the porcine pseudorabies are mainly based on vaccine immunization prevention at home and abroad, and the detection of the pseudorabies virus gE antigen and the gE antibody is combined to eliminate and purify wild virus infected pigs. At present, the PR vaccines clinically applied at home and abroad can be roughly divided into the following three types: firstly, after the separated wild virus or virulent virus is inactivated by formaldehyde, an adjuvant is added for emulsification to prepare an oil emulsion inactivated vaccine; secondly, the separated wild virus or strong virus is repeatedly passaged by non-porcine cells or chick embryos to weaken and develop a low-virulent vaccine; thirdly, the gene deletion vaccine is constructed by utilizing the gene engineering technology. Although safe, the inactivated vaccine has limited immune efficacy and cannot achieve the aim of epidemic disease purification. The safety of the attenuated vaccine has hidden trouble, long production period and high cost. In addition, the gene deletion inactivated vaccine and the whole virus inactivated vaccine for the porcine pseudorabies virus grow on adherent cells, and animal source protein is needed, so that the risk is brought to vaccine manufacture.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a fusion protein of porcine pseudorabies virus, which has good broad spectrum and good immunogenicity, and the prepared antibody has high titer and can prevent various subtypes of porcine pseudorabies virus.

The second object of the present invention is to provide a method for producing the above-mentioned fusion protein, which can realize the expression of the above-mentioned fusion protein of high quality in a large amount.

The third purpose of the invention is to provide the application of the fusion protein.

The fourth purpose of the invention is to provide a porcine pseudorabies virus vaccine containing the fusion protein.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a fusion protein of porcine pseudorabies virus comprises a gB segment and a gD segment;

the gB segment is expressed by a nucleotide sequence shown in SEQ ID NO.1, and the gD segment is expressed by a nucleotide sequence shown in SEQ ID NO. 2.

Further, the gB section and the gD section are arranged in a gB-gD sequence and are connected through a Linker;

the Linker has a nucleotide sequence shown as SEQ ID NO. 3.

Further, the fusion protein is a protein expressed by an HEK293-F cell expression system.

A method for preparing the fusion protein, wherein a gene for coding the fusion protein is expressed in a mammalian expression system.

Further, the gene encoding the fusion protein was expressed in a HEK293-F cell expression system.

Further, providing an expression vector containing a gene for encoding the fusion protein, introducing the expression vector into an HEK293-F cell, then screening the HEK293-F cell to obtain an HEK293-F cell strain for stably expressing the fusion protein, and expressing the HEK293-F cell strain to obtain the fusion protein;

preferably, the screening comprises pressure screening and monoclonal screening.

Further, screening HEK293-F cells expressing the fusion protein using a geneticin screening system;

preferably, the expression vector used in the geneticin screening system is pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13, preferably pcDNA3.1.

The fusion protein or the fusion protein prepared by the preparation method is applied to at least one of the following A) to D):

A) preparing a porcine pseudorabies virus vaccine;

B) preparing an antibody of the porcine pseudorabies virus;

C) preparing a kit for detecting the porcine pseudorabies virus;

D) preparing the porcine pseudorabies virus diagnostic antigen.

A porcine pseudorabies virus vaccine comprising the fusion protein of the porcine pseudorabies virus.

Further, the dosage of the fusion protein in the porcine pseudorabies virus vaccine is 70-90 mu g per head;

preferably, the vaccine further comprises adjuvants including one or more of vaccine adjuvants, stabilizers and antibiotics;

preferably, the vaccine adjuvant comprises aluminium hydroxide gel, freund's complete adjuvant, freund's incomplete adjuvant, white oil adjuvant, carbomer, propolis, MF59 adjuvant or ISA201, preferably ISA201 is used.

Compared with the prior art, the invention has the beneficial effects that:

the fusion protein of the porcine pseudorabies virus provided by the invention comprises a gB segment and a gD segment, wherein the gB segment is expressed by a nucleotide sequence shown in SEQ ID NO.1, and the gD segment is expressed by a nucleotide sequence shown in SEQ ID NO. 2. The sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are sequences obtained by selecting the genes of a classical strain and a current epidemic strain as research objects and performing comparative analysis, and the sequences are optimized and modified by codons, so that the aims of further improving the broad-spectrum property of the fusion protein antigen and improving the antigen expression quantity are fulfilled. The fusion protein has good broad spectrum and good immunogenicity, and the prepared antibody has high titer and can prevent various subtypes of porcine pseudorabies viruses.

The invention provides a preparation method of the fusion protein, which can realize the mass expression of the fusion protein with high quality, has simple operation and is suitable for large-scale production.

The porcine pseudorabies virus vaccine containing the fusion protein provided by the invention has low preparation cost which is less than 1% of the production cost of the existing vaccine, and has good immune effect. After 21-day-old piglets are immunized by the vaccine, blood is respectively taken after 21 days of immunization to measure neutralizing antibodies, the result shows that the neutralizing antibodies in 21 days are not lower than 1:1000, and the challenge result shows that the vaccine can effectively prevent the challenge (JS strain) of classical virulent strains (SC strains) and current epidemic strains, and the protection rate can reach 100%. The vaccine is superior to conventional vaccines.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the result of electrophoresis of PCR amplification products of gB gene of example 1 of the present invention, wherein the lanes from left to right are: marker DL15000, gB gene PCR amplification product;

FIG. 2 shows the result of electrophoresis of the PCR amplification product of gD gene of example 2 of the present invention, wherein the following lanes are shown from left to right: marker DL15000, gD gene PCR amplification product.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

A fusion protein of porcine pseudorabies virus comprises a gB segment and a gD segment, wherein the gB segment is expressed by a nucleotide sequence shown in SEQ ID NO.1, and the gD segment is expressed by a nucleotide sequence shown in SEQ ID NO. 2.

The gB protein is used as a main component formed by the envelope of the PRV, is also an important immunogenic component of the PRV, and can stimulate the organism to generate complement-dependent and complement-independent neutralizing antibodies after entering the organism.

The gD protein is one of essential glycoproteins, which are necessary for PRV invading cells to infect, and the neutralizing antibody induced by gD can perform the neutralizing reaction of PRV regardless of the existence of complement.

The sequences shown in SEQ ID NO.1 and SEQ ID NO.2 are sequences obtained by selecting the genes of a classical strain and a current epidemic strain as research objects and performing comparative analysis, and the sequences are optimized and modified by codons, so that the aims of further improving the broad-spectrum property of the fusion protein antigen and improving the antigen expression quantity are fulfilled. The fusion protein has good broad spectrum and good immunogenicity, and the prepared antibody has high titer and can prevent various subtypes of porcine pseudorabies viruses.

In some embodiments of the invention, the gB segment and the gD segment are arranged in the sequence gB-gD and are linked by a Linker, wherein the Linker has the nucleotide sequence shown in SEQ ID NO. 3. The gB and the gD are connected by a Linker, so that the aim of protein recombination is achieved, and the mutual influence of the formation of respective high-level structures of the gB and gD proteins is avoided. The nucleotide sequence of the encoded fusion protein is shown as SEQ ID NO.4, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 5.

In a preferred embodiment of the invention, the fusion protein is a protein expressed by the HEK293-F cell expression system. The HEK293-F cell is a mammalian expression system, and the HEK293-F cell is used as a host cell, so that the loss of the conformational epitope of the fusion protein is avoided.

A method for preparing the fusion protein, which expresses the gene for coding the fusion protein in a mammalian expression system. The preparation method can realize the mass expression of the high-quality fusion protein, is simple to operate and is suitable for large-scale production. The mammalian expression system may be, for example, but is not limited to, HEK293-F cells, HEK 293-E cells, HEK 293-T cells, CHO cells, or COS cells.

In a preferred embodiment of the invention, the gene encoding the fusion protein is expressed in a HEK293-F cell expression system. HEK293 is a stable cell line obtained after adenovirus Ad5 transfects human embryonic kidney cells, HEK293-F is a derived cell line of HEK293, and has the advantages of easy transfection, high expression, natural glycosylation modification, protein correct folding permission, relevant post-translational modification and the like.

In some embodiments of the present invention, the method for determining the level of a fusion protein of porcine pseudorabies virus comprises: providing an expression vector containing a gene for encoding the fusion protein, introducing the expression vector into an HEK293-F cell, screening the HEK293-F cell to obtain an HEK293-F cell strain for stably expressing the fusion protein, and expressing the HEK293-F cell strain to obtain the fusion protein. Preferably, the expression level of the HEK293-F cell strain for stably expressing the fusion protein is not less than 1 mg/ml.

In a preferred embodiment of the present invention, the screening comprises pressure screening and monoclonal screening. The pressurized screening is firstly utilized to be beneficial to quickly obtaining the cell strain expressing the fusion protein, and then the monoclonal screening is utilized to obtain the high-expression pure cell strain of the fusion protein.

In a preferred embodiment of the invention, a geneticin screening system is used to screen HEK293-F cells expressing the fusion protein. G geneticin (G-148) is an aminosaccharide antibiotic, similar in structure to neomycin, gentamicin, kanamycin, blocks protein synthesis by affecting 80S ribosomal function, is toxic to both prokaryotic and eukaryotic cells, including bacteria, yeast, plant and mammalian cells, and also includes protozoa and helminths. When the neo gene is integrated into the proper place of the eukaryotic cell genome, it can start the transcription of the sequence coded by the neo gene into mRNA, so that the high-efficiency expression of the resistance product aminoglycoside phosphotransferase can be obtained, and the cell can obtain resistance and can grow in the selective culture medium containing G-418 geneticin.

In a preferred embodiment of the present invention, the expression vector used in the geneticin screening system is pcDNA3.1, pEE6.4, pEE12.4 or pGL4.13, preferably pcDNA3.1.

The fusion protein or the fusion protein prepared by the preparation method is applied to at least one of the following A) to D):

A) preparing a porcine pseudorabies virus vaccine;

B) preparing an antibody of the porcine pseudorabies virus;

C) preparing a kit for detecting the porcine pseudorabies virus;

D) preparing the porcine pseudorabies virus diagnostic antigen.

A porcine pseudorabies virus vaccine comprising the fusion protein of the porcine pseudorabies virus. The vaccine has low preparation cost which is less than 1% of the production cost of the existing vaccine, and has good immune effect. After 21-day-old piglets are immunized by the vaccine, blood is respectively taken after 21 days of immunization to measure neutralizing antibodies, the result shows that the neutralizing antibodies in 21 days are not lower than 1:1000, and the challenge result shows that the vaccine can effectively prevent the challenge (JS strain) of classical virulent strains (SC strains) and current epidemic strains, and the protection rate can reach 100%. The vaccine is superior to conventional vaccines.

In some preferred embodiments of the invention, the amount of fusion protein in the porcine pseudorabies vaccine is 70-90. mu.g per head.

In some embodiments of the invention, the vaccine further comprises adjuvants including one or more of vaccine adjuvants, stabilizers, and antibiotics.

In some embodiments of the invention, the vaccine adjuvant comprises alumina hydrate, freund's complete adjuvant, freund's incomplete adjuvant, white oil adjuvant, carbomer, propolis, MF59 adjuvant, or ISA201, preferably ISA201 is used.

In order to facilitate understanding of the technical solutions provided by the present invention, the technical solutions provided by the present invention are further described below with reference to the embodiments.

Example 1 fusion protein sequence encoding porcine pseudorabies Virus

Selecting genes of a classical strain (SC strain) and a current epidemic strain (JS strain) selected from Genebank as research objects, selecting dominant epitopes as components of vaccine antigens through comparative analysis, optimizing and modifying codons of the sequences according to the preference of codons of HEK293-F cells to obtain nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, wherein an agarose gel electrophoresis picture of a PCR (polymerase chain reaction) enriched gB sequence is shown in figure 1, and an agarose gel electrophoresis picture of a PCR enriched gD sequence is shown in figure 2. The middle of gB and gD sequences are connected by a sequence shown in SEQ ID NO.3, so as to further improve the broad spectrum of the fusion protein antigen and improve the antigen expression quantity.

EXAMPLE 2 construction of recombinant vectors expressing fusion proteins

The above synthesized sequence encoding the fusion protein was cloned into the eukaryotic transfer vector pcDNA3.1 by Mlu I and Hind III site insertion. Connecting by T4DNA ligase at 16 ℃ overnight to obtain a connecting product, transforming by escherichia coli competent DH5 alpha, coating the connecting product on an LB plate containing the ampicillin, culturing at 37 ℃ overnight, picking out positive colonies, culturing in an LB culture medium containing the ampicillin, and extracting plasmids. And obtaining the correct recombinant plasmid through PCR, double enzyme digestion and sequencing verification.

Example 3 pressurized selection of recombinant cells

Transfecting the recombinant plasmid to HEK293-F cells which grow well and are suspended completely, carrying out cell passage after 72 hours, adding 600 mu G/ml G418 into a culture medium, pressurizing until the survival rate is about 10%, stopping pressurizing, culturing by using a conventional culture medium, pressurizing and screening once again when the cell survival rate reaches more than 90%, and carrying out next-step screening when the cell survival rate reaches more than 90%.

EXAMPLE 4 monoclonal screening of recombinant cells

Positive clone selection and detection: and (3) selecting the cells growing in an adherent manner into a 96-well plate after culturing for 7 days, culturing for 7 days by utilizing an adherent culture medium, adding 100 mu L of suspension culture medium Freestyle HEK293-F, culturing for 2 days, using the culture medium in the well plate for dot hybridization detection, transferring the high-expression clone into a 24-well plate, culturing for 2 days by utilizing the adherent culture medium, then switching to Freestyle HEK293-F culture, using the culture medium for western blotting detection, and finally obtaining the high-expression clone strain according to the experimental result. All cells were cultured at 37 ℃ in 5% CO₂。

3 cell strains with relatively high expression level are obtained by screening and are respectively named as 293F-gB-gD-1, 293F-gB-gD-2 and 293F-gB-gD-3.

EXAMPLE 5 culture of recombinant cells

Screening HEK293-F cells in Freestyle HEK293Resuspending cells in-F Medium at a cell seeding density of 0.3X 10⁶～0.5×10⁶VC/ml, inoculating cells into a cell shake flask with a proper volume, placing the cell shake flask at 36-38 ℃, and containing 5% CO₂And (5) carrying out shake flask culture in a shaking table for 48-72 hours, and continuously carrying out subculture amplification on the cells.

Performing culture fermentation by using a bioreactor step by step according to needs, generally performing 5-8 times amplification, performing antigen expression when the amplification is carried out to a specific volume, performing culture at 36-38 ℃ to 5 th day, reducing the temperature to 31-33 ℃, adjusting the pH to 7.5 +/-0.1, performing culture at a proper rotating speed, adding 10% of initial working volume of effective Feed at 4 th day and 9 th day, detecting the glucose concentration every day, and supplementing glucose to 3-4 g/L when the glucose concentration is lower than 2.5 g/L. When the cell viability is lower than 80%, the supernatant is harvested to be the required antigen.

By optimizing the culture condition, the feeding time and the feeding amount of the cell strain, the gB-gD protein expression of 1.0-1.2 mg/ml is finally obtained.

Example 6 porcine pseudorabies virus vaccine

Cell debris removal after quantification of expression, vaccine was prepared by mixing with ISA201 adjuvant. Wherein the emulsification ratio of the water phase to the oil phase is 1:1 by mass, the oil phase is firstly led into a beaker, slowly stirred, the water phase is slowly added, after the addition is finished, 10000r/min shearing emulsification is carried out for 5min, 1% thimerosal solution is added according to the total amount before the emulsification is stopped, the final concentration is 0.01%, and the mixture is fully stirred and uniformly mixed. And (4) carrying out sterile quantitative subpackaging, covering and sealing to obtain the porcine pseudorabies virus subunit vaccine, and storing at 2-8 ℃.

After 21-day-old piglets are immunized by the vaccine, blood is respectively taken after 21 days of immunization to measure neutralizing antibodies, the result shows that the neutralizing antibodies in 21 days are not lower than 1:1000, and the challenge result shows that the vaccine can effectively prevent the attack of classical virulent strains (SC strains) and current epidemic strains (JS strains), and the protection rate can reach 100%. All the immunized pigs have no body temperature rise and abnormal clinical symptoms.

Comparative example 1

The classical virulent strain (SC strain) and the current epidemic strain (JS strain) are used for attacking healthy 21-day-old piglets, and the results show that the control pigs have clinical symptoms of the porcine pseudorabies virus, such as the temperature rise of over 41 ℃, and the accompanying neurological symptoms, narcolepsy, vomit, diarrhea and the like.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Tiankang biological products Ltd

Fusion protein of porcine pseudorabies virus, preparation method, application and vaccine thereof

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cgcgaccgct tctacgtgtg cccccccccc agcggcagca ccgtggtgcg cctggagccc 420

gagcaggcct gccccgagta cagccagggc cgcaacttca ccgagggcat cgccgtgctg 480

ttcaaggaga acatcgcccc ccacaagttc aaggcccaca tctactacaa gaacgtgatc 540

gtgaccaccg tgtggagcgg cagcacctac gccgccatca ccaaccgctt caccgaccgc 600

gtgcccgtgc ccgtgcagga gatcaccgac gtgatcgacc gccgcggcaa gtgcgtgagc 660

aaggccgagt acgtgcgcaa caaccacaag gtgaccgcct tcgaccgcga cgagaacccc 720

gtggaggtgg acctgcgccc cagccgcctg aacgccctgg gcacccgcgg ctggcacacc 780

accaacgaca cctacaccaa gatcggcgcc gccggcttct accacaccgg caccagcgtg 840

aactgcatcg tggaggaggt ggaggcccgc agcgtgtacc cctacgacag cttcgccctg 900

agcaccggcg acatcgtgta catgagcccc ttctacggcc tgcgcgaggg cgcccacggc 960

gagcacatcg gctacgcccc cggccgcttc cagcaggtgg agcactacta ccccatcgac 1020

ctggacagcc gcctgcgcgc cagcgagagc gtgacccgca acttcctgcg caccccccac 1080

ttcaccgtgg cctgggactg ggcccccaag acccgccgcg tgtgcagcct ggccaagtgg 1140

cgcgaggccg aggagatgat ccgcgacgag acccgcgacg gcagcttccg cttcaccagc 1200

cgcgccctgg gcgccagctt cgtgagcgac gtgacccagc tggacctgca gcgcgtgcac 1260

ctgggcgact gcgtgctgcg cgaggccagc gaggccatcg acgccatcta ccgccgccgc 1320

tacaacaaca cccacgtgct ggccggcgac aagcccgagg tgtacctggc ccgcggcggc 1380

ttcgtggtgg ccttccgccc cctgatcagc aacgagctgg cccagctgta cgcccgcgag 1440

ctggagcgcc tgggcctggc cggcgtggtg ggccccgcca gccccgccgc cgcccgccgc 1500

gcccgccgca gccccggccc cgccggcacc cccgagcccc ccgccgtgaa cggcaccggc 1560

cacctgcgca tcaccaccgg cagcgccgag ttcgcccgcc tgcagttcac ctacgaccac 1620

atccaggccc acgtgaacga catgctgagc cgcatcgccg ccgcctggtg cgagctgcag 1680

aacaaggacc gcaccctgtg gggcgagatg agccgcctga accccagcgc cgtggccacc 1740

gccgccctgg gccagcgcgt gagcgcccgc atgctgggcg acgtgatggc catcagccgc 1800

tgcgtggagg tgcgcggcgg cgtgtacgtg cagaacagca tgcgcgtgcc cggcgagcgc 1860

ggcacctgct acagccgccc cctggtgacc ttcgagcaca acggcaccgg cgtgatcgag 1920

ggccagctgg gcgacgacaa cgagctgctg atcagccgcg acctgatcga gccctgcacc 1980

ggcaaccacc gccgctactt caagctgggc ggcggctacg tgtactacga ggactacagc 2040

tacgtgcgca tggtggaggt gcccgagacc atcagcaccc gcgtgaccct gaacctgacc 2100

ctgctggagg accgcgagtt cctgcccctg gaggtgtaca cccgcgagga gctggccgac 2160

accggcctgc tggactacag cgagatccag cgccgcaacc agctgcacgc cctgaagttc 2220

tacgacatcg accgcgtggt gaaggtggac cacaacgtgg tgctgctgcg cggcatcgcc 2280

aacttcttcc agggcctggg cgacgtgggc gccgccgtgg gcaaggtggt gctgggcgcc 2340

accggcgccg tgatcagcgc cgtgggcggc atggtgagct tcctgagcaa ccccttcggc 2400

gccctggcca tcggcctgct ggtgctggcc ggcctggtgg ccgccttcct ggcctaccgc 2460

cacatcagcc gcctgcgccg caaccccatg aaggccctgt accccgtgac caccaaggcc 2520

ctgaaggagg acggcgtgga ggaggacgac gtggacgagg ccaagctgga ccaggcccgc 2580

gacatgatcc gctacatgag catcgtgagc gccctggagc agcaggagca caaggcccgc 2640

aagaagaaca gcggccccgc cctgctggcc agccgcgtgg gcgccatggc cacccgccgc 2700

cgccactacc agcgcctgga gaacgaggac cccgacgccc cccctcccag ccccagcctg 2760

ctggccgccc tgctggccgc cctggtggcc cgcaccaccc tgggcgccga cgtggacgcc 2820

gtgcccgccc ccaccttccc cccccccgcc tacccctaca ccgagagctg gcagctgacc 2880

ctgaccaccg tgcccagccc cttcgtgggc cccgccgacg tgtaccacac ccgccccctg 2940

gaggacccct gcggcgtggt ggccctgatc agcgaccccc aggtggaccg cctgctgaac 3000

gaggccgtgg cccaccgccg ccccacctac cgcgcccacg tggcctggta ccgcatcgcc 3060

gacggctgcg cccacctgct gtacttcatc gagtacgccg actgcgaccc ccgccagatc 3120

ttcggccgct gccgccgccg caccaccccc atgtggtgga cccccagcgc cgactacatg 3180

ttccccaccg aggacgagct gggcctgctg atggtggccc ccggccgctt caacgagggc 3240

cagtaccgcc gcctggtgag cgtggacggc gtgaacatcc tgaccgactt catggtggcc 3300

ctgcccgagg gccaggagtg ccccttcgcc cgcgtggacc agcaccgcac ctacaagttc 3360

ggcgcctgct ggagcgacga cagcttcaag cgcggcgtgg acgtgatgcg cttcctgacc 3420

cccttctacc agcagccccc ccaccgcgag gtggtgaact actggtaccg caagaacggc 3480

cgcaccctgc cccgcgccta cgccgccgcc accccctacg ccatcgaccc cgcccgcccc 3540

agcgccggca gcccccgccc ccgcccccgc ccccgccccc gcccccgccc caagcccgag 3600

cccgcccccg ccacccccgc cccccccggc cgcctgcccg agcccgccac ccgcgaccac 3660

gccgccggcg gccgccccac cccccgcccc ccccgccccg agacccccca ccgccccttc 3720

gccccccccg ccgtggtgcc cagcggctgg ccccagcccg ccgagccctt ccccccccgc 3780

accaccgccg cccccggcgt gagccgccac cgcagcgtga tcgtgggcac cggcaccgcc 3840

atgggcgccc tgctggtggg cgtgtgcgtg tacatcttct tccgcctgcg cggcgccaag 3900

ggctaccgcc tgctgggcgg ccccgccgac gccgacgagc tgaaggccca gcccggcccc 3960

taa 3963

<210> 5

<211> 1320

<212> PRT

<213> Artificial sequence

<400> 5

Met Pro Ala Gly Gly Gly Leu Trp Arg Gly Pro Arg Gly His Arg Pro

1 5 10 15

Gly His His Gly Gly Ala Gly Leu Gly Arg Leu Trp Pro Ala Pro His

20 25 30

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

35 40 45

Ala Leu Ala Ala Thr Pro Thr Cys Gly Ala Ala Ala Val Thr Arg Ala

50 55 60

Ala Ser Ala Ser Pro Ala Pro Gly Thr Gly Ala Thr Pro Asp Gly Phe

65 70 75 80

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

85 90 95

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

100 105 110

Ala Val Arg Ala Ala Ala Thr Glu Arg Asp Arg Phe Tyr Val Cys Pro

115 120 125

Pro Pro Ser Gly Ser Thr Val Val Arg Leu Glu Pro Glu Gln Ala Cys

130 135 140

Pro Glu Tyr Ser Gln Gly Arg Asn Phe Thr Glu Gly Ile Ala Val Leu

145 150 155 160

Phe Lys Glu Asn Ile Ala Pro His Lys Phe Lys Ala His Ile Tyr Tyr

165 170 175

Lys Asn Val Ile Val Thr Thr Val Trp Ser Gly Ser Thr Tyr Ala Ala

180 185 190

Ile Thr Asn Arg Phe Thr Asp Arg Val Pro Val Pro Val Gln Glu Ile

195 200 205

Thr Asp Val Ile Asp Arg Arg Gly Lys Cys Val Ser Lys Ala Glu Tyr

210 215 220

Val Arg Asn Asn His Lys Val Thr Ala Phe Asp Arg Asp Glu Asn Pro

225 230 235 240

Val Glu Val Asp Leu Arg Pro Ser Arg Leu Asn Ala Leu Gly Thr Arg

245 250 255

Gly Trp His Thr Thr Asn Asp Thr Tyr Thr Lys Ile Gly Ala Ala Gly

260 265 270

Phe Tyr His Thr Gly Thr Ser Val Asn Cys Ile Val Glu Glu Val Glu

275 280 285

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

290 295 300

Ile Val Tyr Met Ser Pro Phe Tyr Gly Leu Arg Glu Gly Ala His Gly

305 310 315 320

Glu His Ile Gly Tyr Ala Pro Gly Arg Phe Gln Gln Val Glu His Tyr

325 330 335

Tyr Pro Ile Asp Leu Asp Ser Arg Leu Arg Ala Ser Glu Ser Val Thr

340 345 350

Arg Asn Phe Leu Arg Thr Pro His Phe Thr Val Ala Trp Asp Trp Ala

355 360 365

Pro Lys Thr Arg Arg Val Cys Ser Leu Ala Lys Trp Arg Glu Ala Glu

370 375 380

Glu Met Ile Arg Asp Glu Thr Arg Asp Gly Ser Phe Arg Phe Thr Ser

385 390 395 400

Arg Ala Leu Gly Ala Ser Phe Val Ser Asp Val Thr Gln Leu Asp Leu

405 410 415

Gln Arg Val His Leu Gly Asp Cys Val Leu Arg Glu Ala Ser Glu Ala

420 425 430

Ile Asp Ala Ile Tyr Arg Arg Arg Tyr Asn Asn Thr His Val Leu Ala

435 440 445

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

450 455 460

Phe Arg Pro Leu Ile Ser Asn Glu Leu Ala Gln Leu Tyr Ala Arg Glu

465 470 475 480

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

485 490 495

Ala Ala Arg Arg Ala Arg Arg Ser Pro Gly Pro Ala Gly Thr Pro Glu

500 505 510

Pro Pro Ala Val Asn Gly Thr Gly His Leu Arg Ile Thr Thr Gly Ser

515 520 525

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

530 535 540

Val Asn Asp Met Leu Ser Arg Ile Ala Ala Ala Trp Cys Glu Leu Gln

545 550 555 560

Asn Lys Asp Arg Thr Leu Trp Gly Glu Met Ser Arg Leu Asn Pro Ser

565 570 575

Ala Val Ala Thr Ala Ala Leu Gly Gln Arg Val Ser Ala Arg Met Leu

580 585 590

Gly Asp Val Met Ala Ile Ser Arg Cys Val Glu Val Arg Gly Gly Val

595 600 605

Tyr Val Gln Asn Ser Met Arg Val Pro Gly Glu Arg Gly Thr Cys Tyr

610 615 620

Ser Arg Pro Leu Val Thr Phe Glu His Asn Gly Thr Gly Val Ile Glu

625 630 635 640

Gly Gln Leu Gly Asp Asp Asn Glu Leu Leu Ile Ser Arg Asp Leu Ile

645 650 655

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

660 665 670

Tyr Val Tyr Tyr Glu Asp Tyr Ser Tyr Val Arg Met Val Glu Val Pro

675 680 685

Glu Thr Ile Ser Thr Arg Val Thr Leu Asn Leu Thr Leu Leu Glu Asp

690 695 700

Arg Glu Phe Leu Pro Leu Glu Val Tyr Thr Arg Glu Glu Leu Ala Asp

705 710 715 720

Thr Gly Leu Leu Asp Tyr Ser Glu Ile Gln Arg Arg Asn Gln Leu His

725 730 735

Ala Leu Lys Phe Tyr Asp Ile Asp Arg Val Val Lys Val Asp His Asn

740 745 750

Val Val Leu Leu Arg Gly Ile Ala Asn Phe Phe Gln Gly Leu Gly Asp

755 760 765

Val Gly Ala Ala Val Gly Lys Val Val Leu Gly Ala Thr Gly Ala Val

770 775 780

Ile Ser Ala Val Gly Gly Met Val Ser Phe Leu Ser Asn Pro Phe Gly

785 790 795 800

Ala Leu Ala Ile Gly Leu Leu Val Leu Ala Gly Leu Val Ala Ala Phe

805 810 815

Leu Ala Tyr Arg His Ile Ser Arg Leu Arg Arg Asn Pro Met Lys Ala

820 825 830

Leu Tyr Pro Val Thr Thr Lys Ala Leu Lys Glu Asp Gly Val Glu Glu

835 840 845

Asp Asp Val Asp Glu Ala Lys Leu Asp Gln Ala Arg Asp Met Ile Arg

850 855 860

Tyr Met Ser Ile Val Ser Ala Leu Glu Gln Gln Glu His Lys Ala Arg

865 870 875 880

Lys Lys Asn Ser Gly Pro Ala Leu Leu Ala Ser Arg Val Gly Ala Met

885 890 895

Ala Thr Arg Arg Arg His Tyr Gln Arg Leu Glu Asn Glu Asp Pro Asp

900 905 910

Ala Pro Pro Pro Ser Pro Ser Leu Leu Ala Ala Leu Leu Ala Ala Leu

915 920 925

Val Ala Arg Thr Thr Leu Gly Ala Asp Val Asp Ala Val Pro Ala Pro

930 935 940

Thr Phe Pro Pro Pro Ala Tyr Pro Tyr Thr Glu Ser Trp Gln Leu Thr

945 950 955 960

Leu Thr Thr Val Pro Ser Pro Phe Val Gly Pro Ala Asp Val Tyr His

965 970 975

Thr Arg Pro Leu Glu Asp Pro Cys Gly Val Val Ala Leu Ile Ser Asp

980 985 990

Pro Gln Val Asp Arg Leu Leu Asn Glu Ala Val Ala His Arg Arg Pro

995 1000 1005

Thr Tyr Arg Ala His Val Ala Trp Tyr Arg Ile Ala Asp Gly Cys

1010 1015 1020

Ala His Leu Leu Tyr Phe Ile Glu Tyr Ala Asp Cys Asp Pro Arg

1025 1030 1035

Gln Ile Phe Gly Arg Cys Arg Arg Arg Thr Thr Pro Met Trp Trp

1040 1045 1050

Thr Pro Ser Ala Asp Tyr Met Phe Pro Thr Glu Asp Glu Leu Gly

1055 1060 1065

Leu Leu Met Val Ala Pro Gly Arg Phe Asn Glu Gly Gln Tyr Arg

1070 1075 1080

Arg Leu Val Ser Val Asp Gly Val Asn Ile Leu Thr Asp Phe Met

1085 1090 1095

Val Ala Leu Pro Glu Gly Gln Glu Cys Pro Phe Ala Arg Val Asp

1100 1105 1110

Gln His Arg Thr Tyr Lys Phe Gly Ala Cys Trp Ser Asp Asp Ser

1115 1120 1125

Phe Lys Arg Gly Val Asp Val Met Arg Phe Leu Thr Pro Phe Tyr

1130 1135 1140

Gln Gln Pro Pro His Arg Glu Val Val Asn Tyr Trp Tyr Arg Lys

1145 1150 1155

Asn Gly Arg Thr Leu Pro Arg Ala Tyr Ala Ala Ala Thr Pro Tyr

1160 1165 1170

Ala Ile Asp Pro Ala Arg Pro Ser Ala Gly Ser Pro Arg Pro Arg

1175 1180 1185

Pro Arg Pro Arg Pro Arg Pro Arg Pro Lys Pro Glu Pro Ala Pro

1190 1195 1200

Ala Thr Pro Ala Pro Pro Gly Arg Leu Pro Glu Pro Ala Thr Arg

1205 1210 1215

Asp His Ala Ala Gly Gly Arg Pro Thr Pro Arg Pro Pro Arg Pro

1220 1225 1230

Glu Thr Pro His Arg Pro Phe Ala Pro Pro Ala Val Val Pro Ser

1235 1240 1245

Gly Trp Pro Gln Pro Ala Glu Pro Phe Pro Pro Arg Thr Thr Ala

1250 1255 1260

Ala Pro Gly Val Ser Arg His Arg Ser Val Ile Val Gly Thr Gly

1265 1270 1275

Thr Ala Met Gly Ala Leu Leu Val Gly Val Cys Val Tyr Ile Phe

1280 1285 1290

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

1295 1300 1305

Ala Asp Ala Asp Glu Leu Lys Ala Gln Pro Gly Pro

1310 1315 1320

Claims (1)

1. A preparation method of a fusion protein of porcine pseudorabies virus is characterized in that the fusion protein comprises a gB segment and a gD segment;

the gB segment is expressed by a nucleotide sequence shown in SEQ ID NO.1, and the gD segment is expressed by a nucleotide sequence shown in SEQ ID NO. 2;

the gB section and the gD section are arranged in a gB-gD sequence and are connected through a Linker; the Linker is a nucleotide sequence shown as SEQ ID NO. 3;

the preparation method comprises the steps of providing an expression vector pcDNA3.1 containing a gene for encoding the fusion protein, introducing the expression vector into an HEK293-F cell, then carrying out pressure screening and monoclonal screening on the HEK293-F cell to obtain an HEK293-F cell strain stably expressing the fusion protein, culturing at 36-38 ℃ to 5 th day, reducing the temperature to 31-33 ℃, adjusting the pH to 7.5 +/-0.1, culturing, adding 10% of an initial working volume of Effeicient Feed at 4 th day and 9 th day, detecting the glucose concentration every day, and supplementing glucose to 3-4 g/L when the glucose concentration is lower than 2.5 g/L; when the cell viability is lower than 80%, the supernatant is harvested to obtain the fusion protein.

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