CN111978411A

CN111978411A - Porcine reproductive and respiratory syndrome subunit vaccine and preparation method and application thereof

Info

Publication number: CN111978411A
Application number: CN202010789394.9A
Authority: CN
Inventors: 张华伟; 孙芳; 徐高原; 周明光; 曾小燕; 朱娴静; 郝根喜; 邵伟
Original assignee: Wuhan Keqian Biological Co ltd
Current assignee: Wuhan Keqian Biological Co ltd
Priority date: 2020-08-07
Filing date: 2020-08-07
Publication date: 2020-11-24
Anticipated expiration: 2040-08-07
Also published as: CN111978411B

Abstract

The invention relates to the field of genetic engineering and the field of veterinary biopharmaceutics, and particularly discloses a porcine reproductive and respiratory syndrome subunit vaccine and a preparation method and application thereof. The invention firstly provides a fusion protein, the amino acid sequence of which is shown as SEQ ID No.1, and then provides a porcine reproductive and respiratory syndrome subunit vaccine, which comprises the fusion protein. The fusion protein in the porcine reproductive and respiratory syndrome subunit vaccine is obtained by fusion expression of an ORF5 full-length gene of an PRRSV NADC30-like strain subjected to codon optimization and a gene of pseudomonas aeruginosa with a Domain III Domain removed, and by using an insect baculovirus/insect cell expression system. The vaccine has good protection effect, can induce effective humoral immunity and cellular immunity, and is suitable for preventing the blue-ear disease in clinic.

Description

Porcine reproductive and respiratory syndrome subunit vaccine and preparation method and application thereof

Technical Field

The invention relates to the field of genetic engineering and the field of veterinary biopharmaceutics, in particular to a porcine reproductive and respiratory syndrome subunit vaccine and a preparation method and application thereof.

Background

The porcine reproductive and respiratory syndrome is a highly infectious disease caused by PRRSV, can cause sow reproductive disturbance, respiratory symptoms of pigs of various ages, high mortality of piglets and the like, and is one of important diseases causing serious economic loss in the pig industry. The vaccines for preventing the diseases in the market at present mainly comprise inactivated vaccines and attenuated vaccines. The inactivated vaccine has high safety but cannot well induce cellular immunity, and the attenuated vaccine has good immune protection effect but has the risk of virus dispersion and strong return. Whereas subunit vaccines are expected to be able to achieve better prophylactic effects.

The newly-developed epidemic strain NADC30-like can cause the porcine reproductive and respiratory syndrome of swine farms to be attacked, compared with highly-pathogenic PRRSV, the NADC30-like is relatively mild in toxicity, can cause pathogenic symptoms and 30% -50% of death rate of classical strains, has high homology with the American NADC30 strain in the gene sequence, and is also named as NADC 30-like. No related vaccine aiming at the strain exists in the market, and the existing blue-ear disease vaccine cannot play a good role in defending against the blue-ear disease outbreak caused by the strain.

The PRRSV genome comprises 8 open reading frames, and the encoded important structural proteins are mainly ORF5, ORF6, ORF7, which encode GP5 protein (also known as E protein), M protein and N protein, respectively. The GP5 protein is not only capable of inducing both cellular and humoral immune responses. And the surface of the protein is also provided with a neutralization site. However, the GP5 protein is the main envelope glycoprotein of PRRSV and is one of the most obvious proteins in variability, the sequence homology between gene subtypes of European strains and North American strains is only 51% -55%, and even among the same gene subtype strains, the homology is only about 94%. Therefore, the relevant vaccines need to be studied with a focus on different strains.

The existing porcine reproductive and respiratory syndrome vaccine design scheme in the prior art comprises the following steps: "a subunit vaccine capable of inducing an immune response against porcine reproductive and respiratory syndrome virus (application No. 201610333073.1)", "production of a porcine reproductive and respiratory syndrome subunit vaccine (application No. 201310190308.2)", and "a recombinant plasmid, a recombinant viral vector, a recombinant viral strain and use thereof, a recombinant protein and a subunit vaccine containing the protein (application No. 201510257656.6)". The stimulation of the immune response of the cells of the body by vaccines designed according to the thinking of the vaccines still needs to be further improved.

Therefore, further research on the construction of a subunit vaccine against porcine reproductive and respiratory syndrome is necessary.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a vaccine which has good protection effect and is suitable for clinically preventing porcine reproductive and respiratory syndrome.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention firstly provides a fusion protein, and the amino acid sequence of the fusion protein is shown as SEQ ID No. 1.

The invention also provides a coding gene of the fusion protein, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 2. The encoding gene encodes the above fusion protein.

The invention also provides a biological material containing the coding gene, and the biological material comprises recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or engineering bacteria.

The invention also provides an application of the fusion protein or the coding gene or the biological material in the preparation of the porcine reproductive and respiratory syndrome subunit vaccine.

The invention further provides a porcine reproductive and respiratory syndrome subunit vaccine, and the effective component of the porcine reproductive and respiratory syndrome subunit vaccine is the fusion protein.

The invention also provides a preparation method of the porcine reproductive and respiratory syndrome subunit vaccine, which comprises the step of mixing the fusion protein with an adjuvant.

Wherein the preparation method of the fusion protein comprises the following steps:

(1) carrying out codon optimization on the ORF5 full-length gene sequence of the PRRSV NADC30-like strain;

(2) and (3) connecting the gene sequence of the pseudomonas aeruginosa exotoxin A with the Domain III Domain removed with the optimized full-length gene sequence of the ORF5 in series, and adding a His tag and a restriction enzyme site to synthesize the gene sequence of the fusion protein.

The preparation method of the fusion protein further comprises the following steps:

(3) connecting the synthesized gene sequence of the fusion protein into an insect baculovirus expression vector, converting escherichia coli competence, and extracting a recombinant shuttle vector;

(4) transfecting the extracted recombinant shuttle vector into insect cells to obtain a recombinant baculovirus;

(5) inoculating the obtained recombinant baculovirus into insect cells, and harvesting and purifying the target protein.

The nucleotide sequence (ORF5 full-length gene) of GP5 protein adopted in the porcine reproductive and respiratory syndrome subunit vaccine disclosed by the invention is derived from a newly-released epidemic strain PRRSV NADC30-like, and can play a role in preventing outbreak of diseases caused by the strain. The GP5 proteins of different strains show different N glycosylation sites, and the change of the sites can generate the influence on the corresponding immune response which can not be easily expected, so the vaccine design idea of the existing strain can not be simply applied to the development of the vaccine of a new strain, therefore, the invention repeatedly researches the construction of the vaccine of the PRRSV NADC30-like strain aiming at the characteristics of the strain, and finally obtains the targeted vaccine with ideal effect recorded by the invention.

Pseudomonas Exotoxin A (PEA) is a virulence factor for Pseudomonas aeruginosa infection and has 4 functional domains, Domain Ia, Ib, II and III. Wherein, Domain Ia is a cell binding functional region, Domain II is a translocation functional region, and the terminal amino acid sites of Domain Ib and Domain III are ADP ribosylation active regions, i.e. toxicity domains. The invention removes the Domain III toxic structure Domain of PEA (PEA delta DIII) and combines with the specific pathogen immunogenicity region to express recombinant protein (PEA delta DIII-GP 5), and prepares subunit vaccine through purification and emulsification to immunize the organism, thereby stimulating the organism to generate stronger specific cellular immune response and further improving the protection effect of the subunit vaccine.

The subunit vaccine is GP5 full protein sequence, is derived from an epidemic strain PRRSV NADC30-like, and comprises a translocation domain of pseudomonas aeruginosa exotoxin protein PEA, and compared with the subunit vaccine only containing virus antigen protein, the subunit vaccine can better stimulate the humoral immunity and cellular immune response of organisms to PRRSV. The fusion protein is a secretory protein, can be directly secreted out of cells without the processes of posttranslational modification and endoplasmic reticulum retention, and has simpler and more convenient purification process.

Furthermore, the subunit vaccine prepared by the invention contains PRRSV NADC30-like GP5 protein of porcine reproductive and respiratory syndrome virus, a pseudomonas aeruginosa exotoxin translocation domain (PEA delta D III) and a purification tag of 8 XHis.

The preparation of the vaccine specifically comprises the following steps that the genetic engineering technology is utilized to connect the GP5 protein of PRRSV (porcine reproductive and respiratory syndrome virus) epidemic strain NADC30-like and the coding region of Pseudomonas aeruginosa exotoxin protein (toxicity removing structural domain) in series to an insect-baculovirus system expression vector, and the coding region is connected with an insect cell High Five through the insect cell^TM(purchased from Invitrogen) expression, recombinant protein purification and emulsification process, and can be used as vaccine for preventing porcine reproductive and respiratory syndrome.

The invention has the beneficial effects that:

1. compared with a prokaryotic expression system in the prior art, the invention adopts an insect cell expression system, and can maintain the structure and modification state of protein in eukaryotic organisms to the maximum extent.

2. The invention adopts ORF5 full-length gene after codon optimization, does not have carboxyl terminal KEDL signal peptide, and the fusion protein can be secreted out of cells, thus being easier to separate and purify;

3. according to the invention, the recombinant protein formed by fusion expression of ORF5 full-length

gene PEA structures

1 and 2 is adopted, no inflammatory reaction is found after an immune test pig, and the vaccine has good safety;

4. the subunit vaccine disclosed by the invention contains a pseudomonas aeruginosa exotoxin PEA translocation domain capable of stimulating cellular immune response, so that an organism can be effectively stimulated to generate antigen-specific T cell immune response, and the immunogenicity of the subunit vaccine in the scheme is improved;

5. the N end of the subunit vaccine also comprises 8 continuous histidines, namely 8 multiplied by His labels, which is beneficial to the purification step of the fusion protein subunit vaccine;

6. the vaccine has good protection effect, can induce effective humoral immunity and cellular immunity, and is suitable for preventing the blue-ear disease in clinic.

Drawings

FIG. 1 shows the expression results of WB detection of PEA. DELTA.DIII-GP 5, wherein

lanes

1, 2 and 3 show the expression of target proteins in cell culture solutions at days 3, 5 and 8 after HiFi cells were inoculated with recombinant baculovirus rAc-PEA. DELTA.DIII-GP 5, respectively;

FIG. 2 shows the result of detecting the purification of PEA. DELTA.DIII-GP 5 by SDS-PAGE, wherein lane 1 shows the culture of non-inoculated HiFi cells; lane 2 is the culture medium on day 8 after inoculation of HiFi cells with recombinant baculovirus rAc-PEA Δ DIII-GP 5; lane 3 shows the target protein obtained by purifying the culture solution on day 8 after inoculating HiFi cells with recombinant baculovirus rAc-PEA delta D III-GP 5;

FIG. 3 shows the purification results of WB detection of PEA Δ DIII-GP 5, wherein lane 1 shows the test results of a sample of purified fusion protein PEA Δ DIII-GP 5 diluted 50 times;

FIG. 4 is a graph showing the results of the IFN-. gamma.level assay in the serum of the experimental swine on day 14 after the second immunization.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

Example 1

Construction of target protein expression vector

1. Obtaining a target protein sequence:

obtaining gene sequences of PRRSV NADC30-like strains from NCBI (Access: MH651743), selecting nucleotide sequences of GP5 protein and optimizing the nucleotide sequences; obtaining the gene sequence (GenBank: CP039293) of Pseudomonas aeruginosa from NCBI, and selecting the translocation Domain (except Domain III, PEA delta D III) sequence of exotoxin PEA; the gene sequence (shown as SEQ ID No. 1) of the recombinant protein (PEA delta D III-GP 5) is biosynthesized by connecting the PEA delta D III with the optimized GP5 gene sequence in series, adding an 8 XHis tag sequence, and adding NdeI and XhoI enzyme cutting sites at the N end and the C end respectively.

2. The sequence of the synthesized fusion protein pUC-PEA delta D III-GP 5 was ligated into the expression vector pFastBacDual using NdeI and XhoI cleavage sites:

(1) restriction enzyme digestion of pUC-PEA DeltaDIII-GP 5 and insect baculovirus expression vector pFastBacDual (purchased from Fenghui organism, product No. BR 248):

by ddH₂The O content was 40 ul.

Enzyme cutting conditions are as follows: digesting in water bath at 37 ℃ for 1 h.

And (3) carrying out agarose gel electrophoresis after enzyme digestion, cutting and recovering the gel, and operating according to the standard steps of the gel recovery kit.

(2) PEA. DELTA.DIII-GP 5 was attached to pFastBacDual:

connection conditions are as follows: 22 ℃ for 2 h.

Secondly, constructing recombinant baculovirus rAc-PEA delta D III-GP 5

1. Obtaining a recombinant shuttle vector:

transferring 4ul of recombinant transfer plasmid pFastBacdual-PEA delta D III-GP 5 into 100ul of escherichia coli competent DH10Bac (purchased from GiBCO BRL), carrying out ice bath for 30min, then carrying out heat shock at 42 ℃ for 1min, carrying out ice bath for 3min, adding 900ul of nonresistant LB, carrying out resuscitation at 37 ℃ for 4h, coating the solution on a three-antibody (kanamycin, gentamicin and tetracycline) LB plate, carrying out culture at 37 ℃ for 24-48h, screening and purifying a positive bacterial colony through a blue-white spot, and extracting rAc-PEA delta D III-GP 5 of the recombinant rod.

The extraction method comprises the following steps: aseptically picking positive white colony in three-antibody LB liquid culture medium, culturing for 12-16h, collecting thallus, resuspending with 0.3mL solution I (50mmol/L glucose, 10mmol/L EDTA, 25mmol/L Tris-Cl (pH8.0)), adding 0.3mL solution II (0.2mol/L NaOH, 1% SDS), slightly mixing, standing at room temperature for 5min, slowly adding 0.3mL solution III (3mol/L CH)₃COOK, pH5.0), ice bath for 5-10min, centrifuging at 14000r/min for 10min, adding the supernatant into 0.5mL of isopropanol, mixing, ice bath for 5-10min, centrifuging at 14000r/min for 15min, washing the precipitate with 70% ethanol, drying, dissolving in 40 μ L of sterile water, and immediately using or storing at-20 deg.C.

2. Obtaining a recombinant baculovirus:

the extracted recombinant shuttle vector is transfected into Sf9 insect cells (from probiology GmbH of Wuhan family) by using a liposome transfection method, cultured at 27 ℃, cytopathic after 48-72h, and cell culture supernatant is collected to obtain recombinant baculovirus which is immediately used or stored in dark at-80 ℃.

Transfection methods were performed according to the Liposome instructions (lipo2000 from Invitrogen).

Expression and purification of target protein PEA delta DIIII-GP 5

1. Expression of the protein of interest PEA Δ diiii-GP 5:

inoculating the harvested recombinant baculovirus into suspension-cultured insect cells High Five^TM(purchased from Invitrogen) at a virus dose of 0.01MOI and a cell density of 1.0X 10⁶The cell volume was 400 ml/ml. Cell culture supernatants were harvested at 3, 5, and 8 days later, respectively, and Western blotting was performed to detect the expression of the target protein PEA. DELTA.DIII-GP 5, and the detection results are shown in FIG. 1.

2. And (3) purification of the target protein:

the purification method adopts conventional nickel column affinity chromatography. The specific operation steps are as follows: taking High Five 8 days after virus inoculation^TMCentrifuging at 10000rpm to remove cells and cell debris, and filtering with 0.45um filter membrane to remove fine impurities; combining the filtered supernatant with a nickel column and passing the nickel column through the column; washing with a washing buffer (50mM imidazole, 1 XPBS) column washing; elution buffer (150mM imidazole, 1 XPBS) was run through the column; the eluate was dialyzed overnight at 4 ℃ against a dialysis buffer (1 XPBS) to obtain the objective protein. And detecting the purified protein by SDS-PAGE and Western blotting. The results are shown in FIG. 2 and FIG. 3, respectively.

Preparation of four, PEA delta DIII-GP 5 subunit vaccine

The concentration of the purified PEA delta DIII-GP 5 protein is measured, then the protein is filtered, and the protein is emulsified with sterile ISA201 adjuvant (purchased from SEPPIC company) according to the proportion of 1:1 to prepare subunit vaccine, so that the content of PEA delta DIII-GP 5 antigen in each milliliter of vaccine is 200ug, and the subunit vaccine is stored at 4 ℃ for standby. Sterile dialysis buffer (1 × PBS) was fully emulsified with an equal volume of ISA201 adjuvant to prepare a vaccine control, which was placed in a refrigerator at 4 ℃ for future use.

EXAMPLE 1 PEA DeltaDIII-GP 5 subunit vaccine safety testing in mice

10 Balb/C mice of 4-5 weeks old were purchased and divided into 2 groups of 5 mice each, namely an immunization group and a control group. The immunization group was inoculated with 200. mu.L of the PEA delta D III-GP 5 subunit vaccine prepared in example 1 by intramuscular injection of the calf on day 0 and 14, respectively; the blank control group was injected with an equal volume of vaccine control in the same manner. After the continuous observation for 14 days after immunization, the tested mice have normal spirit and appetite, good health condition and no adverse reaction, which indicates that the subunit vaccine has good safety to the mice. The results of the mouse safety test are shown in table 1.

TABLE 1

Experimental example 2 PEA delta DIII-GP 5 subunit vaccine safety and efficacy on piglets

1. The safety and effectiveness of the PEA delta DIII-GP 5 subunit vaccine on piglets are tested:

10 pigs of 45-day-old PRRSV negative piglets were selected and divided into 2 groups, 5 piglets in the immunization group and 5 piglets in the control group. The immunized group was injected with 1.0ml of the PEA delta D III-GP 5 subunit vaccine prepared in example 1 in the retroauricular muscle on day 0 and day 14, respectively, and the blank control group was not immunized. After continuous observation for 14 days, no clinical symptoms appear on piglets, the spirit, appetite and body temperature are normal, no adverse reaction is generated, and the result shows that the subunit vaccine has good safety on the piglets.

The specific results of the piglet safety test are shown in table 2.

TABLE 2

2. Immunopotentiality test for PEA DeltaDIII-GP 5 subunit vaccine

10 PRRSV negative piglets of 45 days old were selected and divided into 2 groups. PEA delta DIIII-GP 5 subunit vaccine immunization group 5 respectively; blank control group 5 heads. The immunized group was given 1.0ml of vaccine by retroauricular muscle injection on day 0 and 14, respectively, and the blank control group was not immunized. Collecting blood from the vena cava before the day of the second immunization (before immunization), 14 th day after the second immunization, 28 th day, 42 th day, 56 th day and 60 th day after the second immunization respectively, and performing ELISA antibody level detection; and IFN-. gamma.levels in serum were measured on day 14 (test kit was purchased from BD Co., BD-EL-P0003, according to the kit instructions) and neutralizing antibody titers. The test results are shown in Table 3, FIG. 4 (porcine serum IFN-. gamma.level test results) and Table 4, respectively. The test result shows that the PEA delta D III-GP 5 subunit vaccine has good immunogenicity and can stimulate the organism to generate specific antibodies with higher level.

The specific results of the piglet effectiveness test (ELISA antibody level detection) are shown in Table 3.

TABLE 3

The ELISA antibody level detection of the experimental example adopts a pig breeding and respiratory syndrome virus ELISA antibody detection kit purchased from Wuhan's Probiotics, Inc., and the specific method is as follows:

the PEA. DELTA.DIII-GP 5 recombinant protein was diluted to 1. mu.g/ml with coating solution, coated on 96-well Elisa plates at 100. mu.l/well and coated overnight at 4 ℃. Adding PBS washing solution 200ul, and repeatedly washing for 2 times; and (3) adding 100 mu l of diluted serum (diluted by serum diluent in the kit according to the kit specification and the ratio of 1: 40) to-be-detected serum and positive (PRRSV specific serum purchased from Chinese veterinary medicine inspection institute) into the hole of the antigen-coated plate respectively, and setting 2 holes for positive control and negative control respectively. Gently shaking and mixing, and incubating at 37 ℃ for 30 minutes. Adding PBS washing solution 200ul, washing repeatedly for 5 times, and finally drying; HRP-labeled anti-pig lgG secondary antibody (used by diluting 1000-fold with PBS) was added to 100. mu.l per well, and incubated at 37 ℃ for 30 minutes. Add PBS wash 200ul, repeat washing 5 times, finally clap to dry. Adding 50 mul of substrate solution A into each hole, adding 50 mul of substrate solution B into each hole, slightly vibrating the reaction plate, mixing uniformly, and developing for 10 minutes at room temperature in a dark place; adding 50 mul of stop solution into each hole, and slightly vibrating the reaction plate to mix uniformly; and setting a measuring wavelength of 630nm by a microplate reader for testing.

The specific method for measuring the titer of the neutralizing antibody is as follows:

(1) serum treatment

Inactivating the sterile serum to be detected in 56 ℃ water bath for 30 min.

(2) Serum dilution

50 μ l of serum-free DMEM medium was added to each well of a 96-well cell culture plate, followed by serial dilutions of the serum to be tested from 1:2 to 1:256, each dilution being repeated 4 times, 50 μ l per well.

(3) Neutralization of viruses

Diluting PRRSV NADC30-like strain virus solution with TCID50 to 100 μ l of virus solution containing 400 TCID50 with serum-free DMEM medium, adding diluted virus solution into serum dilution well, placing 50 μ l each well at 37 deg.C, and adding 5% CO₂The incubator of (1) was allowed to act for 1 hour.

(4) Addition of Marc-145 cells

After digesting full monolayer Marc-145 cells with pancreatin, blowing down with DMEM medium (containing 4% newborn calf serum), counting, adjusting cell density to 2.0 x 10 with DMEM medium (containing 4% newborn calf serum)⁵The adjusted cell suspension was inoculated into neutralized 96-well cell culture plates at a concentration of 100. mu.l/well and at 37 ℃ in 5% CO₂The incubator of (2) for cultivation.

(5) Cell controls

8-well cell controls were set up in the above 96-well cell culture plates, and 100. mu.l of serum-free DMEM medium was added to each well.

(6) Determination of results

Cytopathic (CPE) was observed and recorded daily for 4 consecutive days.

And (3) calculating the titer of PRRSV neutralizing antibodies in the detected serum according to a Reed-Muench method when the cells correspond to the cell-free lesion. The results are shown in Table 4.

TABLE 4

Table 4 above shows the results of neutralization tests with PRRSV NADC30-like from sera collected on day 14 after the second immunization of test pigs. Therefore, the vaccine of the invention has high average titer and ideal immune effect.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

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Claims

1. A fusion protein is characterized in that the amino acid sequence of the fusion protein is shown as SEQ ID No. 1.

2. The encoding gene of the fusion protein is characterized in that the nucleotide sequence of the encoding gene is shown as SEQ ID No. 2.

3. Biological material comprising the encoded gene of claim 2, said biological material comprising recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors or engineered bacteria.

4. Use of the fusion protein of claim 1 or the coding gene of claim 2 or the biomaterial of claim 3 in the preparation of a porcine reproductive and respiratory syndrome subunit vaccine.

5. The porcine reproductive and respiratory syndrome subunit vaccine is characterized in that the effective component of the porcine reproductive and respiratory syndrome subunit vaccine is the fusion protein of claim 1.

6. The method of preparing a porcine reproductive and respiratory syndrome subunit vaccine of claim 5, comprising the step of mixing the fusion protein of claim 1 with an adjuvant.

7. The method according to claim 6, wherein the method for producing the fusion protein comprises:

8. The method of claim 7, wherein the method further comprises: