CN114457104A - Expression vector of porcine pseudorabies virus glycoprotein gD and preparation method and application thereof - Google Patents

Abstract

The invention discloses an expression vector of porcine pseudorabies virus glycoprotein gD, a preparation method and application thereof, wherein the downstream of a promoter contains an expression plasmid of PRV-gD gene; the promoter is a constitutive promoter H36, the gene sequence of the constitutive promoter H36 is shown as SEQ ID No.4, and the gene sequence of PRV-gD is shown as SEQ ID No. 2. The PRV-gD expressed by the invention has immunological activity, and provides technical guidance and materials for the research of the porcine pseudorabies subunit vaccine and the establishment of a high-efficiency immunological detection technology.

Description

Expression vector of porcine pseudorabies virus glycoprotein gD and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an expression vector of porcine pseudorabies virus glycoprotein gD, and a preparation method and application thereof.

Background

Porcine Pseudorabies virus (PRV) is a virulent infectious disease which causes various domestic animals, poultry and wild animals to suffer from fever, extreme itching (except pigs) and encephalomyelitis as main symptoms, has wide host range and is mostly lethal infection, and causes huge loss to the pig industry all over the world.

Studies have shown that the PRV-gD glycoprotein, also known as gp50, which has a total of 402 amino acid residues and a molecular weight of approximately 45kD, is one of the major envelope glycoproteins on the surface of mature PRV virions, and is also an essential structural protein of PRV upon invasion into host cells, and plays an important role in the infection and proliferation of PRV. The gD has high homology among different PRV strains, high conservation and good immunogenicity, and can be used as an ideal antigen for serological detection.

The corynebacterium glutamicum can produce correctly folded foreign proteins due to no endotoxin production and low extracellular hydrolase activity, and can be used as an excellent host for fermentation production of various foreign proteins, particularly for medicinal proteins with strict limitations on endotoxin and the like.

Disclosure of Invention

This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.

The present invention has been made keeping in mind the above and/or other problems occurring in the prior art.

One of the purposes of the invention is to provide an expression vector of porcine pseudorabies virus glycoprotein gD, the constructed prokaryotic recombinant expression plasmid PRV-gD is induced and compositely expressed, and the product is detected by SDS-PAGE and Western-Blot, and the result shows that PRV-gD protein can perform specific reaction with PRV-gD antibody positive serum, can be used as a diagnostic antigen for detecting PRV serum antibody, and lays a foundation for further developing a PRV antibody ELISA detection method.

In order to solve the technical problems, the invention provides the following technical scheme: an expression vector of porcine pseudorabies virus glycoprotein gD, which contains an expression plasmid of PRV-gD gene at the downstream of a promoter;

wherein, the promoter is a constitutive promoter H36.

As a preferable embodiment of the expression vector of the porcine pseudorabies virus glycoprotein gD of the present invention, wherein: the gene sequence of the constitutive promoter H36 is shown in SEQ ID NO. 4.

As a preferable embodiment of the expression vector of the porcine pseudorabies virus glycoprotein gD of the present invention, wherein: the PRV-gD gene sequence is shown as SEQ ID NO. 2.

As a preferable embodiment of the expression vector of the porcine pseudorabies virus glycoprotein gD of the present invention, wherein: the starting vector for constructing the expression vector was pXMJ 19.

As a preferable embodiment of the expression vector of the porcine pseudorabies virus glycoprotein gD of the present invention, wherein: the expression vector is the vector H36-PRV-gD shown in figure 5.

Another object of the present invention is to provide a method for preparing an expression vector of porcine pseudorabies virus glycoprotein gD, comprising,

constructing a target gene expression vector pXMJ19-PRV-gD by using a corynebacterium glutamicum expression plasmid pXMJ 19;

based on pXMJ19-PRV-gD plasmid, double enzyme digestion is carried out through enzyme digestion sites EcoR V and Hind III, and the product is homologously recombined with constitutive promoter H36 to obtain constitutive target gene expression vector H36-PRV-gD.

As a preferred scheme of the preparation method of the expression vector of the porcine pseudorabies virus glycoprotein gD, the invention comprises the following steps: the specific method for constructing the target gene expression vector pXMJ19-PRV-gD by using the corynebacterium glutamicum expression plasmid pXMJ19 comprises the following steps,

taking the plasmid pXMJ19-EGFP as a vector, and obtaining a linear vector fragment pXMJ19 through enzyme digestion, wherein the sequence of the linear vector fragment is shown as SEQ ID NO. 1;

carrying out codon optimization on a target gene, adding Hind III 5 'and EcoRI enzyme cutting sites 3' to synthesize a target gene PRV-gD, wherein the sequence of the target gene is shown as SEQ ID NO. 2;

the linear vector fragment pXMJ19 and the target gene PRV-gD were added in a molar ratio of 1:3, carrying out ligation under the condition, and transforming the Escherichia coli JM109 with the ligation product to obtain a transformant;

and selecting a single colony of a transformant with correct colony PCR and sequencing, culturing, and extracting a plasmid to obtain the target gene expression plasmid pXMJ 19-PRV-gD.

As a preferred scheme of the preparation method of the expression vector of the porcine pseudorabies virus glycoprotein gD, the invention comprises the following steps: the product is homologously recombined with a constitutive promoter H36 by the specific method comprising,

based on pXMJ19-PRV-gD plasmid, double restriction is carried out by restriction enzyme cutting sites EcoR V and Hind III, and the sequence of the restriction enzyme cutting product is shown as SEQ ID NO. 3.

Taking pEC-H36 plasmid as a template, obtaining H36 by PCR, wherein the sequence of the H36 is shown as SEQ ID NO. 4;

homologous recombination is carried out on the enzyme digestion product fragment and the H36 fragment, and the Escherichia coli JM109 is transformed by the ligation product to obtain a transformant;

selecting a single colony of a transformant with correct colony PCR and sequencing, culturing, extracting plasmids, and obtaining a plasmid H36-PRV-gD for constitutive expression of PRV-gD after transfer culture of a target gene expression plasmid.

Another purpose of the invention is to provide an engineering bacterium constructed by the expression vector of the porcine pseudorabies virus glycoprotein gD.

Another purpose of the invention is to provide the application of the engineering bacteria in expressing porcine pseudorabies virus glycoprotein gD.

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

the invention realizes the intracellular soluble expression of porcine pseudorabies virus glycoprotein gD in corynebacterium glutamicum, and the expression quantity is obviously improved after a constitutive promoter is replaced. After PRV-gD positive antibody specificity detection, the PRV-gD expressed by the invention has immunological activity, and provides technical guidance and materials for the research of porcine pseudorabies subunit vaccine and the establishment of high-efficiency immunological detection technology.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:

FIG. 1 is a map of a Corynebacterium glutamicum pXMJ19-PRV-gD expression vector of the present invention.

FIG. 2 is an electrophoretogram of the vector prepared in example 1 of the present invention after enzyme cleavage; wherein, L1, L2 and L3 are biological parallel samples.

FIG. 3 is a diagram showing the PCR results of colonies after ligation of an expression vector and a target gene in example 1 of the present invention; wherein 1-7 are biological repeats.

FIG. 4 is a SDS-PAGE picture after induction of expression of the objective strain in example 3 of the present invention; wherein 1-3 are no-load strain whole bacteria in sequence, supernatant is obtained after crushing, and precipitation is obtained after crushing; 4-6 are all strains of the target strain in sequence, and supernatant is obtained after crushing, and precipitation is obtained after crushing.

FIG. 5 is a WB diagram showing the target strain in example 3 of the present invention after induced expression; wherein 1-3 are no-load strain whole bacteria in sequence, supernatant is obtained after crushing, and precipitation is obtained after crushing; 4-6 are all strains of the target strain in sequence, and supernatant is obtained after crushing, and precipitation is obtained after crushing.

FIG. 6 is a genetic map of the replacement constitutive promoter H36 in example 4 of the present invention.

FIG. 7 is a diagram showing the result of sequencing verification after homologous recombination of a target gene vector and an H36 promoter in example 4 of the present invention; wherein 1-6 are biological repeats.

FIG. 8 is a WB diagram showing the expression of a target strain in example 4 of the present invention; wherein 1 is a supernatant obtained after crushing the no-load strain; 2 is the supernatant of the disrupted inducible expression strain pXMJ 19-gD; 3. 4, the crushed whole strain is a constitutive strain H36-gD, and the crushed supernatant is obtained.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below with reference to examples of the specification.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.

Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.

Example 1: construction of a plasmid for expression of a target Gene

The objective of this example was to obtain a corynebacterium glutamicum expression plasmid for porcine pseudorabies virus glycoprotein gD.

The method comprises the following steps:

(1) plasmid pXMJ19-EGFP is used as a vector, and a linear vector fragment pXMJ19, namely the fragment 1, is obtained by enzyme digestion and nucleic acid electrophoresis separation gel recovery of a large fragment, wherein the sequence is shown as SEQ ID No. 1. The cleavage reaction system is shown in Table 1.

TABLE 1 digestion reaction System

The restriction enzymes are Hind III and EcoR I; the enzyme digestion reaction conditions are as follows: 30min at 37 ℃.

Performing agarose gel electrophoresis on the product after enzyme digestion, recovering an agarose gel band with correct molecular weight, and determining the concentration for later use; the electrophoretogram of the enzyme-digested product is shown in FIG. 2, and the band size is correct.

(2) Through literature research, the amino acid sequence of the target gene is obtained from NCBI, codon optimization is carried out by referring to Corynebacterium glutamicum ATCC13032, Hind III is added at 5 ', EcoRI enzyme cutting sites are added at 3', and the target gene is synthesized by Jinwei to obtain a fragment 2, wherein the sequence is shown as SEQ ID NO. 2.

(3) Fragment 1 and fragment 2 in a molar ratio of 1:3, the ligation was performed overnight at a reaction temperature of 16 ℃ as shown in Table 2. Ligation product was transformed into E.coli JM109 and coated with LBB (Cm)^r) The plate was incubated overnight at 37 ℃ to obtain transformants. Colony PCR and sequencing verification were performed using sequencing primers YZ-gD-S (SEQ ID NO.5) and YZ-gD-A (SEQ ID NO.6), and the results are shown in FIG. 3, with the correct band size.

Selecting single colony of transformant with correct colony PCR and sequencing, and performing PCR sequencing on the single colony at Cm^rThe resistant LBB liquid culture medium is cultured overnight at 37 ℃, and plasmids are extracted to obtain the target gene expression plasmid pXMJ19-PRV-gD, and the map is shown in figure 1.

TABLE 2 enzyme Linked systems

Example 2: fermentative expression of a protein of interest

The purpose of this example is to express a protein of interest by fermentation, as follows:

electrotransformation of the gene expression plasmid of interest into the competent Corynebacterium glutamicum, coating Cm^rResistant LBHis plate, 30 ℃ after 30h incubation, picking single colony to Cm^rResistant LBB medium, cultured overnight at 30 ℃ with initial OD₆₀₀0.2 to Cm^rResistant LBB medium, cultured for 3-5 h (OD)₆₀₀Approximately equal to 0.6-0.8), 1Inducing with IPTG (100mM), culturing at 30 deg.C for 24h, fermenting and expressing.

The specific method for the electric transformation of the corynebacterium glutamicum comprises the following steps:

(1) placing competent cells on ice, adding precooled plasmid (5 μ L) or ligation product, flicking tube wall to mix well, ice-cooling for 15 min;

(2) adding into a precooled 0.1cm electric shock cup, carrying out electric shock twice at 1.8kv for 5ms, adding a recovery medium (1mL LBHis), uniformly mixing, and carrying out water bath at 46 ℃ for 6 min.

(3)30℃，100rpm，3h。

(4)10000rpm, 2min, collect the thallus. Most of the supernatant was aspirated, and 100. mu.L of the culture broth was applied to LBHis plate containing chloramphenicol, and the plate was incubated overnight at 30 ℃ and 200rpm, to allow transformants to grow in about 36 hours.

Example 3: verification of target protein

The purpose of this example is to validate a protein of interest, comprising the following steps:

(1) determining OD of 24h fermentation product₆₀₀The bacterial solution was collected at 8000rpm, centrifuged at 8000rpm, washed twice with PBS, resuspended in PBS, disrupted, and the whole strain was collected, supernatant after disruption, and precipitated after disruption to prepare a protein sample (5 × loading buffer). The crushing conditions are as follows: power 25%, time 3min, pulse/interval 1s/2 s.

(2) And (3) carrying out SDS-PAGE and Western-Blot detection on the prepared protein sample, wherein the antibody is a 6 XHis antibody, and verifying the expression of the target protein. ELISA: the primary antibody is PRV-gD positive serum, the secondary antibody is horse radish peroxide goat anti-pig IgG, and the activity of the target protein is verified. The results of SDS-PAGE and Western-Blot are shown in FIGS. 4 and 5. pXMJ19-PRV-gD can be mostly expressed in Corynebacterium glutamicum in an intracellular manner, but the expression level is low, and no obvious band can be seen by SDS-PAGE while a clear band can be seen by WB.

Wherein, the specific conditions and steps of SDS-PAGE detection are as follows:

reagents required for SDS-PAGE:

1) 30% acrylamide solution (5X)

2) Separating gel buffer solution: 1.5M Tris-HCl (pH 8.8), 0.4% SDS

Weighing Tris 18.2g, adding 50ml water, adjusting pH to 8.8 with 1M hydrochloric acid, adding 0.4g SDS, and finally fixing volume to 100ml with distilled water.

3) Concentrating the gel buffer: 1.0M Tris-HCl (pH 6.8), 0.4% SDS

12.1g of Tris was weighed, 50ml of water was added, pH6.8 was adjusted with 1M hydrochloric acid, 0.4g of SDS was added, and finally, volume was made 100ml with distilled water.

4) 10% Ammonium Persulfate (AP)

5) TEMED (tetramethyl ethylene diamine)

6)5 Loading buffer

SDS (0.1g) + DTT (0.78g) + bromophenol blue (0.05g) + Glycerol (5.0ml) +1M Tris-HCl (2.5ml) with pH6.8, and finally distilled water was added to make the volume to 10 ml.

7)5 electrophoresis buffer:

tris was weighed at 3.0g × 5 ═ 15g, glycine at 18.4g × 5 ═ 92g, SDS 1g × 5 ═ 5g was added, and the solution was dissolved in distilled water to a volume of 1000 ml.

The split glue formulation (10ml, amount of two pieces of glue) is shown in table 3.

TABLE 3

The gel buffer (4ml, amount of two gels) was concentrated as shown in Table 4.

TABLE 4

SDS-PAGE procedures:

(1) and (5) checking whether the rubber plate is clean, and wiping the rubber plate with 75% alcohol to dry if the rubber plate has stains.

(2) And clamping the rubber plate, and detecting the leakage by using water after the bottom of the rubber plate is leveled.

(3) Adding the formulas according to the amount of liquid (generally adding water first), adding TEMED last, mixing, and rapidly adding into the rubber plate, approximately adding to the 3/4 position, taking care that no air bubbles exist, and sealing with water or isopropanol and flattening.

(4) Removing the upper layer of water after the gel is solidified, sucking the water by using a filter paper strip, adding the prepared concentrated gel into a rubber plate, and inserting a comb.

(5) After the upper layer is solidified, the comb is horizontally pulled up. The gel plate was placed in the electrophoresis chamber and 1 × electrophoresis buffer was added.

(6) The treated sample was added and electrophoresed at 120V for 1.5h until the bromophenol blue band ran off the bottom of the plate.

(7) After the glue running is finished, carefully prying off the glue plate, immersing the glue in water, heating to boil, keeping for 30-60s, and then placing on a shaking table to shake for 5 min.

(8) Removing water, adding dye solution, heating to boil, maintaining for 30-60s, and shaking on shaking table for 5 min.

(9) Discarding the dye solution, adding decolorization solution, heating to boil, maintaining for 30-60s, and shaking on shaking table for 5 min.

(10) The gel imaging system takes pictures.

The specific conditions and steps of Western-Blot detection are as follows:

reagents required for Western-Blot:

(1)10 transfer Buffer (5L)

145g of Glycine, 290g of Tris and 18.5g of SDS are fully stirred and dissolved, deionized water is added to the solution to fix the volume to 4L, and then the solution is stored at room temperature. Before use, 80mL of the solution was diluted to 800mL, and 200mL of methanol was added to 1 × transfer Buffer.

(2)TBST Buffer(5L)

44g NaCl and 100mL 1M Tris-HCl (pH 8.0) are fully stirred and dissolved, 2.5mL Tween20 is added and fully mixed, deionized water is added to fix the volume of the solution to 5L, and the solution is stored at 4 ℃.

(3) Blocking buffer

5% skimmed milk powder (W/V) is dissolved in TBST and stored at 4 ℃ for further use.

Western-Blot procedure: comprises the steps of membrane transferring, sealing, antibody hatching and color development imaging.

Film transfer:

(1) and cutting off the glue blocks in the region where the target protein is located from the protein glue according to the protein marker, and soaking the glue blocks in the transfer Buffer.

(2) 6 pieces of filter paper cut in advance are soaked in a transfer Buffer.

(3) And (3) cutting a PVDF film slightly larger than the rubber block, activating the PVDF film in methanol, respectively activating the front surface and the back surface for 10s, shaking the box during activation to enable the PVDF film to fully contact the methanol, and transferring the PVDF film to a transfer Buffer after the activation is finished.

(4) Wetting the plane of the electrotransfer instrument with the roller, putting three pieces of filter paper, and expelling bubbles with the roller.

(5) Placing a PVDF film, and using a roller to drive out bubbles; placing a rubber block, paying attention to the up, down, left and right sides, and slightly driving out the rubber block by using tweezers if bubbles exist; and placing three layers of filter paper, slightly rolling by using a roller, and coating a wet electrotransport instrument cover plate.

(6) The cover plate is closed, the electric resolver switch is opened, the constant current is set to be 0.300A, and the time (the specific membrane conversion time is determined according to the size of the target protein, the larger the molecular weight of the target protein is, the longer the required membrane conversion time is, the smaller the molecular weight of the target protein is, and the shorter the required membrane conversion time is.)

And (3) sealing:

(1) after the film is completely rotated, the protein film is immediately placed in a pre-prepared TBST, and the moisture preservation of the film and the front and back of the film (a corner mark can be cut off in a small part) are required to be noticed from all steps after the film is completely rotated, so that the film is prevented from being dried, and otherwise, a higher background is easily generated.

(2) TBST was washed once, shaken on a side-shaking table for 5 minutes at room temperature, decanted into blocking solution, shaken slowly on a shaking table, and blocked for 60 minutes at room temperature. For some higher background antibodies, blocking may be performed overnight at 4 ℃.

(3) After being sealed, the rubber is cleaned by TBST

Primary antibody incubation:

(1) the primary antibody is diluted with milk in appropriate proportions according to the instructions for the primary antibody.

(2) The diluted primary antibody was immediately added and incubated on a side-shaking shaker at room temperature for 1h with slow shaking. If the primary antibody is not effective after 1h incubation, the incubation can be carried out overnight at 4 ℃ with slow shaking.

(3) Primary antibody was recovered and stored at 4 deg.C (available within one week).

(4) TBST three times, each time in room temperature in the side rocking bed washing membrane 5-10 minutes.

Color development imaging:

(1) ECL color developing solution 1:1 (can cover the whole film)

(2) The film is placed in the central frame of the plate, the whole film is uniformly soaked by the color developing solution, after the film is focused clearly, the luminous image is clicked for shooting, manual shooting/automatic shooting can be selected, single shooting/multiple shooting can be performed, multiple pieces of film can be shot and then directly stored, and the white light image can also be stored by clicking superposition.

Example 4: PRV-gD expression optimization by replacing constitutive promoter H36

This example provides a method for constructing a plasmid for constitutive expression of H36-PRV-gD protein, comprising the following steps:

(1) based on pXMJ19-PRV-gD plasmid, double enzyme digestion is carried out through enzyme digestion sites EcoR V and Hind III, a product gel is recovered to obtain a fragment 3, and the sequence is shown as SEQ ID NO. 3.

(2) pEC-H36 plasmid is used as a template, H36 is obtained through PCR, a fragment 4 is obtained, the nucleotide sequence is shown as SEQ ID NO.4, 20bp homologous sequences are arranged at two ends of the fragment 4 and the fragment 3, and design primers are H36_ F (SEQ ID NO.7) and H36_ R (SEQ ID NO. 8).

(3) And carrying out homologous recombination on the fragment 3 and the fragment 4, wherein the homologous recombination conditions are shown in Table 5, the concentration ratio is required to be 1:3, and the reaction is carried out for 1h at 50 ℃. The transformant was obtained by transforming Escherichia coli JM109 with the ligation product, plating an LBB (Cmr) plate, and culturing overnight at 37 ℃. Colony PCR and sequencing verification were performed using the sequencing primers YZ-H36-S (SEQ ID NO.9) and YZ-H36-A (SEQ ID NO.10), and the band sizes were correct as shown in FIG. 7. Single colonies of transformants with correct colony PCR and sequencing were picked and tested at Cm^rThe resistant LBB liquid culture medium is cultured overnight at 37 ℃, plasmids are extracted, the objective gene expression plasmids are obtained and are subjected to transfer culture to obtain the plasmids H36-PRV-gD of constitutive expression PRV-gD, and the map is shown in figure 6.

TABLE 5 homologous recombination

(4) The H36-PRV-gD was electrotransferred to Corynebacterium glutamicum by the same method as examples 2 and 3, and qualitative verification was carried out, and SDS-PAGE and Western-Blot results are shown in FIG. 8, and constitutive expression vector H36-PRV-gD was expressed in Corynebacterium glutamicum in a higher amount than inducible expression vector pXMJ 19-PRV-gD.

The constructed prokaryotic recombinant expression plasmid PRV-gD is induced and compositely expressed, intracellular soluble expression of porcine pseudorabies virus glycoprotein gD is realized in corynebacterium glutamicum, and the product is detected by SDS-PAGE and Western-Blot, and the result shows that PRV-gD protein can perform specific reaction with PRV-gD antibody positive serum and can be used as a diagnostic antigen for detecting PRV serum antibody. After replacing the constitutive promoter, the expression quantity is obviously improved. After PRV-gD positive antibody specificity detection, PRV-gD expressed in the research has immunological activity, and provides technical guidance and materials for the research of porcine pseudorabies subunit vaccine and the establishment of high-efficiency immunological detection technology.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Sequence listing

<110> university of south of the Yangtze river

<120> expression vector of porcine pseudorabies virus glycoprotein gD and preparation method and application thereof

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ctcattagcg acacgttgcg cagcggccac gtccttagcc ttatccacgc aatcgagaac 2880

gtactgccta accgcgaaat cagactgaat cagtttccaa tcatcgggct tcaccaaagc 2940

aacagcaacg cgggttgatt cgacccgttc cggtgcttcc agaccggcga gcttgtacag 3000

ttcttcttcc atttcacgac gtacatcagc gtctatgtaa tcaatgccca aagcacgctt 3060

agccccacgt gaccaggacg aacgcaggtt tttagaacca acctcatact cacgccaccg 3120

agccaccaaa acagcgtcca tatcctcgcc ggcgtcgctt tgatcggcca acatatccaa 3180

catctgaaac ggcgtgtacg accccttaga cgcggtttta gtagcggagc cagtcagttc 3240

ctgagacatg cccttagcga ggtaggttgc cattttcgca gcgtctccac cccaggtaga 3300

cacctgatca agtttgaccc cgtgctcacg cagtggcgcg tccataccgg ccttaaccac 3360

accagcagac cagcgggaaa acatggaatc ctcaaacgcc ttgagttcat cgtcagacag 3420

tggacgatcc aagaacaaca gcatgttgcg gtgcaagtgc caaccgttcg cccaagagtc 3480

tgtgacctca tagtcactat aggtgtgctc caccccgtac cgtgcacgtt ctttcttcca 3540

ctgagatgtt ttcaccatcg aagagtacgc agtcttaata cccgcttcaa cctgcgcaaa 3600

tgactgtgag cggttgtgtc gaacagtgcc cacaaacatc atgagcgcgc cacccgccgc 3660

caagtgattc ttagtagcaa tagccagctc aatgcggcgt tcgcccatga cttccaattc 3720

agccagaggt gacccccagc gagagtgaga gttttgcaga ccctcaaact gcgaagcacc 3780

gttagacgac caggacaccg caacagcttc gtccctgcgc cacctatggc accccgccag 3840

agccttacta ttggtgatct tgtacatgac gttttgccta cgccacgccc tagcgcgagt 3900

gaccttagaa ccctcattga cctgcggttc cttagaggtg ttcacttcta tttcagtgtt 3960

actcagtgtt acctagaccc gatgttgtgc ggggttgcgc agtgcgagtt tgtgcgggtg 4020

ttgtgcccgt tgtcttagct agtgctatgg ttgtcaattg aaaccccttc gggttatgtg 4080

gcccccgtgc atatgagttg gtagctcgca cgggggtttg tcttgtctag ggactattaa 4140

tttttagtgg tgtttggtgg ccgcctagct tggctatgcg tgccagctta cccgtactca 4200

atgttaaaga tttgcatcga catgggaggg ttacgtgtcc gatacctagg gggggtatcc 4260

gcgactaggt gccccggtgc tcactgtctg taccggcggg gcaagcccca caccccgcat 4320

ggacagggtg gctccgcccc ctgcaccccc agcaatctgc atgtacatgt tttacacatt 4380

agcacgacat gactgcatgt gcatgcactg catgcagact aggtaaatat gagtatgtac 4440

gactagtaac aggagcactg cacataatga atgagttgca ggacaatgtt tgctacgcat 4500

gcgcatgaca tatcgcagga aagctactag agtcttaaag catggcaacc aaggcacagc 4560

tagaacagca actacaagaa gctcaacagg cactacaggc gcagcaagcg caggcacaag 4620

ccaccatcga agcactagaa gcgcaggcaa aggctaagcc cgtcgtggtc accgcacgcg 4680

ttcctttggc actacgtgag gacatgaagc gcgcaggcat gcagaacggt gaaaacctcc 4740

aagagttcat gatcgccgcg tttaccgagc ggctagaaaa gctcaccacc accgacaacg 4800

aggaaaacaa tgtctaaccc actagttctc tttgcccacc gtgacccggt aaatgacgtg 4860

acgttcgagt gcattgagca cgccacctac gacacacttt cacacgctaa agaccagatc 4920

accgcccaaa tgcaagccct agacgaagaa gccgccctac tgccctaatg ggtgtttcat 4980

gggtgtttcc ctagtgtttc atggtgtttt cacctaagct agggaattgc gcgagaagtc 5040

tcgcaaaaat cagcaacccc cggaaccaca cagttcacgg gggttcttct atgccagaaa 5100

tcagaaaggg gaaccagtga acgaccccga atggctggat gatcctccag cgcggggatc 5160

tcatgctgga gttcttcgcc caccccaaaa ggatctaggt gaagatcctt tttgataatc 5220

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 5280

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 5340

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 5400

cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 5460

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 5520

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 5580

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 5640

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcat tgagaaagcg 5700

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 5760

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 5820

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 5880

ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 5940

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 6000

gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 6060

caaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 6120

tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6180

ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6240

taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 6300

tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 6360

aaaagagttt gtagaaacgc aaaaaggcca tccgtcagga tggccttctg cttaatttga 6420

tgcctggcag tttatggcgg gcgtcctgcc cgccaccctc cgggccgttg cttcgcaacg 6480

ttcaaatccg ctcccggcgg atttgtccta ctcaggagag cgttcaccga caaacaacag 6540

ataaaacgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcagtt 6600

ccctactctc gcatggggag accccacact accatcggcg ctacggcgtt tcacttctga 6660

gttcggcatg gggtcaggtg ggaccaccgc gctactgccg ccaggcaaat tctgttttat 6720

cagaccgctt ctgcgttctg atttaatctg tatcaggctg aaaatcttct ctcatccgcc 6780

aaaacagcca agctgaattc 6800

<210> 2

<211> 1233

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aagcttatgc tgctggcagc actgctggca gcactggtcg cacgcaccac cctgggtgca 60

gatgtggatg cagtgcctgc accaaccttc cctccacctg catacccata caccgaatcc 120

tggcagctga ccctgaccac cgtgccatcc ccattcgtgg gccctgccga tgtgtaccac 180

acccgcccac tggaagatcc atgcggcgtg gtggcactga tctccgatcc acaagtggat 240

cgcctgctga acgaagcagt ggcacaccgc cgcccaacct accgcgcaca cgtggcatgg 300

taccgcatcg cagatggctg cgcacacctg ctgtacttca tcgaatacgc agattgcgat 360

ccacgtcaga tcttcggccg ctgccgccgt cgcaccaccc caatgtggtg gaccccatcc 420

gcagattaca tgttcccaac cgaagatgaa ctgggcctgc tgatggtggc acctggccgc 480

ttcaacgaag gtcagtaccg ccgcctggtg tccgtggatg gcgtgaacat cctgaccgat 540

ttcatggtgg cactgcctga aggccaagaa tgcccttttg cacgcgtgga tcagcaccgc 600

acctacaagt tcggcgcatg ctggtccgat gattccttca agcgcggcgt ggatgtgatg 660

cgcttcctga ccccattcta tcagcagcca ccacaccgcg aagtggtgaa ctactggtac 720

cgcaagaacg gccgcaccct gccacgcgca tacgcagccg caaccccata cgcaatcgat 780

cctgcacgcc catccgccgg ctccccacgc ccacgcccac gcccacgccc acgcccaaag 840

cctgaacctg cacctgcaac ccctgcacca cctggccgcc tgcctgaacc tgcaactcgt 900

gatcacgccg ccggcggccg cccaacccca cgcccaccac gccctgaaac cccacaccgc 960

ccattcgcac caccagcagt ggtgccatcc ggctggccac agccagcaga acctttccca 1020

ccacgcacca ccgcagcacc tggcgtctcc cgccaccgct ccgtgatcgt gggcaccggc 1080

accgcaatgg gcgcactgct ggtgggcgtg tgcgtgtaca tcttcttccg cctgcgcggc 1140

gcaaagggct accgcctgct gggcggccca gcagatgcag atgaactgaa ggcacagcct 1200

ggcccacacc atcaccatca ccactaagaa ttc 1233

<210> 3

<211> 6594

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aagcttatgc tgctggcagc actgctggca gcactggtcg cacgcaccac cctgggtgca 60

gatgtggatg cagtgcctgc accaaccttc cctccacctg catacccata caccgaatcc 120

tggcagctga ccctgaccac cgtgccatcc ccattcgtgg gccctgccga tgtgtaccac 180

acccgcccac tggaagatcc atgcggcgtg gtggcactga tctccgatcc acaagtggat 240

cgcctgctga acgaagcagt ggcacaccgc cgcccaacct accgcgcaca cgtggcatgg 300

taccgcatcg cagatggctg cgcacacctg ctgtacttca tcgaatacgc agattgcgat 360

ccacgtcaga tcttcggccg ctgccgccgt cgcaccaccc caatgtggtg gaccccatcc 420

gcagattaca tgttcccaac cgaagatgaa ctgggcctgc tgatggtggc acctggccgc 480

ttcaacgaag gtcagtaccg ccgcctggtg tccgtggatg gcgtgaacat cctgaccgat 540

ttcatggtgg cactgcctga aggccaagaa tgcccttttg cacgcgtgga tcagcaccgc 600

acctacaagt tcggcgcatg ctggtccgat gattccttca agcgcggcgt ggatgtgatg 660

cgcttcctga ccccattcta tcagcagcca ccacaccgcg aagtggtgaa ctactggtac 720

cgcaagaacg gccgcaccct gccacgcgca tacgcagccg caaccccata cgcaatcgat 780

cctgcacgcc catccgccgg ctccccacgc ccacgcccac gcccacgccc acgcccaaag 840

cctgaacctg cacctgcaac ccctgcacca cctggccgcc tgcctgaacc tgcaactcgt 900

gatcacgccg ccggcggccg cccaacccca cgcccaccac gccctgaaac cccacaccgc 960

ccattcgcac caccagcagt ggtgccatcc ggctggccac agccagcaga acctttccca 1020

ccacgcacca ccgcagcacc tggcgtctcc cgccaccgct ccgtgatcgt gggcaccggc 1080

accgcaatgg gcgcactgct ggtgggcgtg tgcgtgtaca tcttcttccg cctgcgcggc 1140

gcaaagggct accgcctgct gggcggccca gcagatgcag atgaactgaa ggcacagcct 1200

ggcccacacc atcaccatca ccactaagaa ttcagcttgg ctgttttggc ggatgagaga 1260

agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 1320

tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 1380

gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 1440

caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 1500

gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa 1560

cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag 1620

aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tatttttcta 1680

aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1740

ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1800

ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1860

agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1920

tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttgctt cctcgctcac 1980

tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 2040

aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 2100

gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 2160

ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 2220

ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 2280

gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg 2340

ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 2400

cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 2460

cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 2520

gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 2580

aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 2640

tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 2700

gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 2760

tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 2820

gatcttcacc tagatccttt tggggtgggc gaagaactcc agcatgagat ccccgcgctg 2880

gaggatcatc cagccattcg gggtcgttca ctggttcccc tttctgattt ctggcataga 2940

agaacccccg tgaactgtgt ggttccgggg gttgctgatt tttgcgagac ttctcgcgca 3000

attccctagc ttaggtgaaa acaccatgaa acactaggga aacacccatg aaacacccat 3060

tagggcagta gggcggcttc ttcgtctagg gcttgcattt gggcggtgat ctggtcttta 3120

gcgtgtgaaa gtgtgtcgta ggtggcgtgc tcaatgcact cgaacgtcac gtcatttacc 3180

gggtcacggt gggcaaagag aactagtggg ttagacattg ttttcctcgt tgtcggtggt 3240

ggtgagcttt tctagccgct cggtaaacgc ggcgatcatg aactcttgga ggttttcacc 3300

gttctgcatg cctgcgcgct tcatgtcctc acgtagtgcc aaaggaacgc gtgcggtgac 3360

cacgacgggc ttagcctttg cctgcgcttc tagtgcttcg atggtggctt gtgcctgcgc 3420

ttgctgcgcc tgtagtgcct gttgagcttc ttgtagttgc tgttctagct gtgccttggt 3480

tgccatgctt taagactcta gtagctttcc tgcgatatgt catgcgcatg cgtagcaaac 3540

attgtcctgc aactcattca ttatgtgcag tgctcctgtt actagtcgta catactcata 3600

tttacctagt ctgcatgcag tgcatgcaca tgcagtcatg tcgtgctaat gtgtaaaaca 3660

tgtacatgca gattgctggg ggtgcagggg gcggagccac cctgtccatg cggggtgtgg 3720

ggcttgcccc gccggtacag acagtgagca ccggggcacc tagtcgcgga taccccccct 3780

aggtatcgga cacgtaaccc tcccatgtcg atgcaaatct ttaacattga gtacgggtaa 3840

gctggcacgc atagccaagc taggcggcca ccaaacacca ctaaaaatta atagtcccta 3900

gacaagacaa acccccgtgc gagctaccaa ctcatatgca cgggggccac ataacccgaa 3960

ggggtttcaa ttgacaacca tagcactagc taagacaacg ggcacaacac ccgcacaaac 4020

tcgcactgcg caaccccgca caacatcggg tctaggtaac actgagtaac actgaaatag 4080

aagtgaacac ctctaaggaa ccgcaggtca atgagggttc taaggtcact cgcgctaggg 4140

cgtggcgtag gcaaaacgtc atgtacaaga tcaccaatag taaggctctg gcggggtgcc 4200

ataggtggcg cagggacgaa gctgttgcgg tgtcctggtc gtctaacggt gcttcgcagt 4260

ttgagggtct gcaaaactct cactctcgct gggggtcacc tctggctgaa ttggaagtca 4320

tgggcgaacg ccgcattgag ctggctattg ctactaagaa tcacttggcg gcgggtggcg 4380

cgctcatgat gtttgtgggc actgttcgac acaaccgctc acagtcattt gcgcaggttg 4440

aagcgggtat taagactgcg tactcttcga tggtgaaaac atctcagtgg aagaaagaac 4500

gtgcacggta cggggtggag cacacctata gtgactatga ggtcacagac tcttgggcga 4560

acggttggca cttgcaccgc aacatgctgt tgttcttgga tcgtccactg tctgacgatg 4620

aactcaaggc gtttgaggat tccatgtttt cccgctggtc tgctggtgtg gttaaggccg 4680

gtatggacgc gccactgcgt gagcacgggg tcaaacttga tcaggtgtct acctggggtg 4740

gagacgctgc gaaaatggca acctacctcg ctaagggcat gtctcaggaa ctgactggct 4800

ccgctactaa aaccgcgtct aaggggtcgt acacgccgtt tcagatgttg gatatgttgg 4860

ccgatcaaag cgacgccggc gaggatatgg acgctgtttt ggtggctcgg tggcgtgagt 4920

atgaggttgg ttctaaaaac ctgcgttcgt cctggtcacg tggggctaag cgtgctttgg 4980

gcattgatta catagacgct gatgtacgtc gtgaaatgga agaagaactg tacaagctcg 5040

ccggtctgga agcaccggaa cgggtcgaat caacccgcgt tgctgttgct ttggtgaagc 5100

ccgatgattg gaaactgatt cagtctgatt tcgcggttag gcagtacgtt ctcgattgcg 5160

tggataaggc taaggacgtg gccgctgcgc aacgtgtcgc taatgaggtg ctggcaagtc 5220

tgggtgtgga ttccaccccg tgcatgatcg ttatggatga tgtggacttg gacgcggttc 5280

tgcctactca tggggacgct actaagcgtg atctgaatgc ggcggtgttc gcgggtaatg 5340

agcagactat tcttcgcacc cactaaaagc ggcataaacc ccgttcgata ttttgtgcga 5400

tgaatttatg gtcaatgtcg cgggggcaaa ctatgatggg tcttgttgtt ggcgtcccgg 5460

aaaacgattc cgaagcccaa cctttcatag aaggcggcgg tggaatcgaa atctcgtgat 5520

ggcaggttgg gcgtcgcttg gtcggtcatt tcgaagggca ccaataactg ccttaaaaaa 5580

attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga 5640

catggaagcc atcacagacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt 5700

cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg 5760

ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat 5820

tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg 5880

aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg 5940

tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct 6000

caccgtcttt cattgccata cggaactccg gatgagcatt catcaggcgg gcaagaatgt 6060

gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa 6120

tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat 6180

gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca 6240

ttttagcttc cttagctcct gaaaatctcg tcgaagctcg gcggatttgt cctactcaag 6300

ctgatccgac aaaatccaca cattatccca ggtgtccgga tcggtcaaat acgctgccag 6360

ctcatagacc gtatccaaag catccggggc tgatccccgg cgccagggtg gtttttcttt 6420

tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca 6480

gcaagcggtc cacgtggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg 6540

cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatc 6594

<210> 4

<211> 229

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgcctccac accgctcgtc acatcctgca aaagctgggt acctctatct ggtgccctaa 60

acgggggaat attaacgggc ccagggtggt cgcaccttgg ttggtaggag tagcatggga 120

tccaagctgt caccggatgt gctttccggt ctgatgagtc cgtgaggacg aaacagcctc 180

tacaaataat tttgtttaat actagagaaa gaggagaaat actagatgc 229

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

catcggctcg tataatgtgt gg 22

<210> 6

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gaccccacac taccatcgg 19

<210> 7

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gtcgtatccc actaccgaga tatcatgcct ccacaccgct 40

<210> 8

<211> 58

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gtgctgccag cagcataagc ttctagtatt tctcctcttt ctctagtatt aaacaaaa 58

<210> 9

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctggccctga gagagttg 18

<210> 10

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cgaatgggga tggcacg 17

Claims (10)

1. An expression vector of porcine pseudorabies virus glycoprotein gD, which is characterized in that: an expression plasmid containing a PRV-gD gene downstream of the promoter;

wherein, the promoter is a constitutive promoter H36.

2. The expression vector of porcine pseudorabies virus glycoprotein gD of claim 1, characterized in that: the gene sequence of the constitutive promoter H36 is shown in SEQ ID NO. 4.

3. The expression vector of porcine pseudorabies virus glycoprotein gD according to claim 1 or 2, characterized in that: the PRV-gD gene sequence is shown as SEQ ID NO. 2.

4. The expression vector of porcine pseudorabies virus glycoprotein gD according to claim 3, characterized in that: the starting vector for constructing the expression vector was pXMJ 19.

5. The expression vector according to any one of claims 1, 2 and 4, comprising the sequence of SEQ ID NO: the expression vector is the vector H36-PRV-gD shown in figure 6.

6. The method for producing an expression vector for porcine pseudorabies virus glycoprotein gD according to any of claims 1 to 5, wherein: comprises the steps of (a) preparing a mixture of a plurality of raw materials,

constructing a target gene expression vector pXMJ19-PRV-gD by using a corynebacterium glutamicum expression plasmid pXMJ 19;

based on pXMJ19-PRV-gD plasmid, double enzyme digestion is carried out through enzyme digestion sites EcoR V and Hind III, and the product is homologously recombined with a constitutive promoter H36.

7. The method for preparing an expression vector for porcine pseudorabies virus glycoprotein gD according to claim 6, wherein: the method for constructing the target gene expression vector pXMJ19-PRV-gD by the corynebacterium glutamicum expression plasmid pXMJ19 comprises the following steps,

taking the plasmid pXMJ19-EGFP as a vector, and obtaining a linear vector fragment pXMJ19 through enzyme digestion, wherein the sequence of the linear vector fragment is shown as SEQ ID NO. 1;

carrying out codon optimization on a target gene, adding Hind III 5 'and EcoRI enzyme cutting sites 3' to synthesize a target gene PRV-gD, wherein the sequence of the target gene is shown as SEQ ID NO. 2;

the linear vector fragment pXMJ19 and the target gene PRV-gD were added in a molar ratio of 1:3, carrying out ligation under the condition, and transforming the Escherichia coli JM109 with the ligation product to obtain a transformant;

and selecting a single colony of a transformant with correct colony PCR and sequencing, culturing, and extracting a plasmid to obtain the target gene expression plasmid pXMJ 19-PRV-gD.

8. The method for producing the expression vector for porcine pseudorabies virus glycoprotein gD according to claim 6 or 7, wherein: the product is homologously recombined with a constitutive promoter H36 by the specific method comprising,

based on pXMJ19-PRV-gD plasmid, double restriction is carried out by restriction enzyme cutting sites EcoR V and Hind III, and the sequence of the restriction enzyme cutting product is shown as SEQ ID NO. 3.

Taking pEC-H36 plasmid as a template, obtaining H36 by PCR, wherein the sequence of the H36 is shown as SEQ ID NO. 4;

homologous recombination is carried out on the enzyme digestion product fragment and the H36 fragment, and the Escherichia coli JM109 is transformed by the ligation product to obtain a transformant;

selecting a single colony of a transformant with correct colony PCR and sequencing, culturing, extracting plasmids, and obtaining a plasmid H36-PRV-gD for constitutive expression of PRV-gD after transfer culture of a target gene expression plasmid.

9. An engineered bacterium constructed from the expression vector of porcine pseudorabies virus glycoprotein gD according to any one of claims 1 to 5.

10. The use of the engineered bacterium of claim 9 to express porcine pseudorabies virus glycoprotein gD.

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