CN106148358A - A kind of method utilizing escherichia expression system to prepare pig parvoviral virus sample particle subunits vaccine and application - Google Patents

Abstract

The invention discloses a gene encoding porcine parvovirus VP2 protein, a method for prokaryotically expressing the VP2 protein virus-like particle and its application in vaccine preparation. Optimize the sequence, artificially synthesize the VP2 gene, insert the synthesized gene into the pET28a vector, and then co-transform the VP2 protein and the chaperone protein into the BL21(DE3) host strain to promote the correct folding of the VP2 protein . Experiments have proved that the VP2 protein expressed by the recombinant bacteria can realize self-assembly in vitro and has good immunogenicity. Using the virus-like particle subunit vaccine prepared by the VP2 protein expressed by the present invention to immunize mice and guinea pigs can induce high levels of hemagglutination-inhibiting antibodies and neutralizing antibodies, and the vaccine can prevent guinea pigs from being infected by porcine parvovirus . The recombinant bacterium of the invention can efficiently prepare porcine parvovirus virus-like particles, has low production cost, simple operation and better biological safety.

Description

Preparation of subunits of porcine parvovirus virus-like particles using Escherichia coli expression system Methods and applications of bit vaccines

technical field

The invention belongs to the field of bioengineering, and specifically relates to a method for soluble expression of porcine parvovirus VP2 protein, an encoding gene, a preparation method of porcine parvovirus virus-like particles, and the application of the virus-like particles as a subunit vaccine.

Background technique

Porcine parvovirus (PPV) is one of the main causes of reproductive disorders in pigs. Its main characteristics are abortion, stillbirth, mummification of primiparous sows and death of newborn piglets. Since Mayr and Mahnel discovered and confirmed the existence and pathogenicity of PPV in 1966, scholars at home and abroad have conducted extensive and in-depth research on it. In recent years, PPV infection has been expanding and rising, which has caused huge economic losses to the global pig industry. In my country, Pan Xuezhu first isolated PPV in 1983. Since then, several strains of PPV have been isolated successively in various places. Although different isolates belong to the same serotype, they can cause various clinical symptoms, and all of them mainly cause reproductive disorders. In addition, porcine parvovirus is often co-infected with other viruses, such as porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus, resulting in multisystem syndrome, dermatitis, and respiratory syndrome in weaned piglets. At present, the PPV positive rate of multiparous sows and breeding boars in my country is as high as 80%.

PPV belongs to the Parvoviridae family and the genus Parvovirus. The genome of PPV is single-stranded negative-strand DNA, which encodes three structural proteins: VP1, VP2, and VP3, and three nonstructural proteins: NS1, NS2, and NS3. The VP2 protein is the main capsid protein of the virus, which has the ability to self-assemble into virus-like particles (virus-like particle, VLP), is a good antigen transporter, and also has good immunogenicity, which can effectively induce The animal's body mounts an immune response.

Many studies and clinical practice have shown that vaccine immunization is a very effective method to control the prevalence of PPV. After immunization, pigs mainly resist PPV infection through humoral immune response. The vaccines used to prevent PPV in my country are mainly inactivated vaccines. Although PPV inactivated vaccines can effectively protect pigs from PPV infection, they have disadvantages such as high production costs, incomplete virus inactivation, and unstable immune effects; In addition, inactivating agents and residual viral DNA may be dangerous to immunized pigs. At present, the new PPV virus-like particle subunit vaccine has been extensively studied. The virus-like particle formed by the VP2 protein has a structure similar to that of the PPV virus particle, and does not contain the genetic material of the virus. Present antigens in natural conformation and pattern, effectively stimulate humoral and cellular immunity, and induce a good immune response in pigs.

At present, insect cell expression systems are mainly used to express PPV virus-like particles in research reports, but their production costs are relatively high. In contrast, the prokaryotic expression system has high protein expression, low production cost, and simple production operation. However, in actual operation, most proteins form inclusion bodies due to incorrect folding, which brings obstacles to subsequent research work and application.

Contents of the invention

One of the objectives of the present invention is to provide a DNA sequence of the artificially modified and synthesized porcine parvovirus VP2 gene, as shown in SEQ ID NO.1.

The second object of the present invention is to provide a recombinant vector expressing porcine parvovirus VP2 protein. After the recombinant vector is introduced into Escherichia coli, it can express VP2 protein in a soluble manner. hemagglutination.

The third object of the present invention is to provide a recombinant bacterium expressing porcine parvovirus VP2 protein, the recombinant bacterium contains VP2 recombinant plasmid and chaperone protein particle, can express VP2 protein solublely, and the VP2 protein has hemagglutination and excellent antigenicity , can be used to prepare porcine parvovirus vaccine.

The fourth object of the present invention is to provide a method for soluble expression of porcine parvovirus VP2 protein using Escherichia coli, which is low in production cost, easy to implement and suitable for large-scale production.

The fifth object of the present invention is to provide a condition for the assembly of porcine parvovirus VP2 protein in vitro, and the virus-like particles formed under the condition have the size, shape and hemagglutination similar to natural parvovirus.

The sixth object of the present invention is to provide the application of a porcine parvovirus VP2 protein in the preparation of a subunit vaccine, which can induce a specific immune response in the body after immunization of animals, and has strong anti-PPV characteristics.

In order to achieve the above object, the present invention takes the following technical measures:

Design a recombinant bacterium expressing porcine parvovirus VP2 protein, and prepare through the following main steps:

(1) Artificial modification and synthesis of VP2 gene

Referring to the VP2 gene sequence of the Chinese strain of porcine parvovirus (GenBank: AY583318), according to the codon preference of Escherichia coli, the complete porcine parvovirus VP2 gene was artificially synthesized, and its nucleotide sequence is shown in SEQ ID NO.1.

(2) Construction of recombinant vector

After the VP2 gene of porcine parvovirus was synthesized, it was amplified by PCR, inserted into the pET28a vector, and the ligated product was transformed into JM109 cells, and the kanamycin-resistant monoclonal was screened, identified by PCR and enzyme digestion, and recombinant pET28a was obtained - VP2 vector.

(3) Construction of recombinant bacteria

Transform the pET28a-VP2 vector into BL21 (DE3) cells, screen for kanamycin-resistant single clones, and carry out PCR identification to obtain recombinant bacteria containing the VP2 expression vector; then (by CaCl ₂ -MgCl ₂ method ) to make the recombinant bacteria competent, and transfer the chaperone protein particle to obtain the recombinant bacteria finally expressing the soluble VP2 protein.

Determination of VP2 protein assembly conditions in vitro: After the induced expression of VP2 protein was purified by affinity chromatography, it was dialyzed at 4°C to remove imidazole and then assembled. By changing the salt ion concentration of the dialysate, the best conditions for VP2 protein assembly in vitro were explored Under optimal conditions, the dialyzed protein was detected by electron microscopy, dynamic light scattering and hemagglutination experiments.

The application of the above-mentioned porcine parvovirus virus-like particles in the preparation of subunit vaccines shows that the vaccine can induce a specific immune response in the body after immunizing animals, and has the property of resisting PPV virulent attack. For the specific solution, see the specific implementation manner.

The beneficial technical effect of the present invention is:

The present invention proposes for the first time the co-expression of chaperone protein and PPV VP2 protein in Escherichia coli, the solubility of the expressed VP2 protein has been significantly improved, and the VP2 protein can self-assemble into VLP under suitable conditions, and the vaccine immunized with the VLP Mice and guinea pigs can induce good specific immune responses and can be used to produce vaccines against porcine parvovirus.

(1) According to the codon preference of Escherichia coli and combined with a large number of practical researches, the present invention artificially modified and synthesized the DNA sequence of porcine parvovirus VP2 gene, and the VP2 protein expressed by using this sequence has good immunogenicity.

(2) In the present invention, the porcine parvovirus VP2 gene is inserted into the pET28a vector and co-expressed with the chaperone protein, which increases the expression of soluble VP2 protein; the selected molecular chaperone can not only prevent the protein from forming aggregates or no The active structure increases the correct folding rate and may also affect the folding path.

(3) The porcine parvovirus VP2 gene expressed in the present invention can form high-quality porcine parvovirus virus-like particles.

(4) The porcine parvovirus virus-like particles expressed in the present invention can be used to prepare subunit vaccines, which can induce specific immune responses against porcine parvovirus after immunizing animals.

Description of drawings

Figure 1: The vector map of pET28a;

The pET28a vector is driven by a T7 promoter with a kanamycin resistance gene.

Figure 2: The enzyme digestion identification map of the prokaryotic expression vector pET28a-VP2;

M: DL2000 DNA Marker; 1&2: pET28a-VP2 bacterial liquid PCR; 3&4: pET28a-VP2 double digestion.

Figure 3: Schematic diagram of analyzing VP2 protein expression for SDS-PAGE;

M: protein marker; 1: induced by BL21(DE3) empty bacteria; 2: induced by pET28a empty vector; 3: supernatant induced by pET28a-VP2 recombinant vector; 4: precipitate induced by pET28a-VP2 recombinant vector; 5: pET28a - Supernatant after co-expression of VP2 recombinant vector and chaperone protein; 6: precipitation after co-expression of pET28a-VP2 recombinant vector and chaperone protein.

Figure 4: Schematic diagram of VP2 protein purification for SDS-PAGE analysis;

M: protein marker; 1: induced by BL21(DE3) empty bacteria; 2: induced by pET28a empty vector; 3: supernatant after co-expression of pET28a-VP2 recombinant vector and chaperone protein; 4: pET28a-VP2 recombinant vector and chaperone protein Products were purified after co-expression.

Figure 5: Schematic diagram of VP2 protein purification for Western-blot analysis;

1: After pET28a empty vector induction; 2: Supernatant after co-expression of pET28a-VP2 recombinant vector and chaperone protein; 3: Purified product after co-expression of pET28a-VP2 recombinant vector and chaperone protein.

Figure 6: Schematic diagram of the electron microscope detection results of VP2 protein assembly in vitro;

After the purified VP2 protein was dialyzed against different buffer solutions, it was negatively stained with phosphotungstic acid and then observed with a transmission electron microscope.

Figure 7: Schematic diagram of the dynamic light scattering detection results of VP2 protein assembly in vitro;

After the purified VP2 protein was dialyzed against different buffers, it was detected with a Malvern Nano-ZS nanometer particle size analyzer.

Figure 8: Schematic diagram of the detection results of hemagglutination activity of VP2 protein assembled in vitro;

After the purified VP2 protein was dialyzed against different buffers, its hemagglutination activity was detected with 0.8% mouse red blood cells.

detailed description

The present invention can be better understood from the following examples. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention. The technical solutions described in the present invention, unless otherwise specified, are conventional solutions in the art, and the reagents, unless otherwise specified, are commercial or published reagent materials.

Example 1

The invention discloses a prokaryotic method for expressing porcine parvovirus VP2 protein. In the method, PPV VP2 protein and chaperone protein are co-expressed to obtain a recombinant expression protein with good solubility and high activity. The specific steps are as follows:

1.1 Construction of recombinant vector pET28a-VP2

1.1.1 Artificial modification and synthesis of PPV main immunogenic gene VP2

Referring to the VP2 gene sequence of PPV China strain (GenBank: AY583318), the sequence was optimized according to the codon preference of Escherichia coli, and the VP2 gene was artificially synthesized, and its nucleotide sequence is shown in SEQ ID NO.1.

1.1.2 Construction of pET28a-VP2 vector

The primer sequences used to amplify the VP2 protein-encoding gene are as follows:

F: 5'- GGATCC ATGTCGGAAAATGTGGAACA-3' ( Bam HI)

R: 5'- AAGCTT ATACAGTTTCCGTGGAATGA-3' ( Hind III)

Among them, the forward primer carries a Bam HI restriction site; the reverse primer carries a Hind III restriction site. Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

Using the above primers, the synthetic VP2 gene was used as a template for PCR amplification. The reaction conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1 min; a total of 30 cycles; Extend at 72°C for 10 min. The amplified gene fragment was detected by 1% agarose gel electrophoresis, and the result is shown in Figure 2. The size of the target gene was about 1700bp, which was consistent with the expected result; the amplified fragment was recovered with Tiangen Gel Recovery Kit, and the specific operation See the manual, the recovered product and the pET28a plasmid were digested with restriction endonucleases Bam HI and Hind III respectively, and the reaction conditions were 37°C for 4 hours.

Subsequently, the pET28a vector and the double-digestion products of the VP2 gene were recovered separately, and then ligated with T4 ligase overnight at 16°C. The ligation product was transformed into Escherichia coli JM109 (TaKaRa) competent cells. For details, please refer to the kit instruction manual. Since the pET28a vector has a kanamycin resistance gene, spread the transformed cells on an LB agar plate containing 50 µg/mL kanamycin resistance. A single colony on the plate was picked randomly, inoculated in LB medium with kanamycin resistance, and cultured at 37°C for 12h. A positive clone was obtained by bacterial liquid PCR and enzyme digestion (the results are shown in Figure 2). The positive plasmid was named pET28a-VP2, and the recombinant plasmid pET28a-VP2 was sent to the company for sequencing. The results showed that the sequence of the inserted fragment was correct, without mutation and inserted The direction is correct.

1.2 Construction and expression of PPV VP2 Escherichia coli expression strain

1.2.1 Construction of PPV VP2 Escherichia coli expression strain

Transform Escherichia coli BL21(DE3) (TaKaRa) competent cells with the positive pET28a-VP2 plasmid obtained in 1.1 above, spread the transformed product on LB agar plate containing 50 μg/mL kanamycin resistance, pick a single colony, After bacterial liquid PCR identification, the screened positive clones were the strains containing recombinant expression plasmids.

In order to realize the co-expression of VP2 protein and chaperone protein to increase the expression of soluble VP2 protein, inoculate the above-mentioned recombinant bacteria in 50 mL LB medium containing kanamycin, culture at 37°C until the OD value is 0.6, and then 4°C , centrifuge at 5000rpm for 5min to collect the bacterial pellet, make it competent by CaCl ₂ -MgCl ₂ method, prepare competent cells, and then transfer the chaperone protein particle into it, so that the BL21(DE3) cells contain the pET28a-VP2 expression plasmid , and contains a chaperone protein plasmid. Since the two plasmids are compatible, the transcription and translation will not affect each other, so the co-expression of VP2 protein and chaperone protein can be achieved. Namycin and 25 μg/mL chloramphenicol on LB agar plates, pick a single colony, which is the recombinant strain co-expressing VP2 protein and chaperone protein.

1.2.2 Expression and identification of prokaryotic expression strains

Inoculate the above-mentioned PPV VP2 expression strain 1:100 into liquid LB medium containing 50 µg/mL kanamycin and 25 µg/mL chloramphenicol, add 50 ng/mL tetracycline to induce the expression of chaperone protein, and culture at 37 °C When the OD value was about 0.6, IPTG (isopropylthiogalactopyranoside induction) was added at a final concentration of 1.0 mmol/L to induce the expression of VP2 protein, and the cells were collected by centrifugation after induction at 25°C for 12 hours.

According to the wet weight of the above bacteria, add buffer A (50mmol/L PB, 250mmol/LNaCl, pH7.0) at a ratio of 1:10 (W/V) to resuspend the bacteria, and then place it on ice for ultrasonic lysis, 12000rpm Centrifuge for 20 min, separate the supernatant and precipitate, then take samples, and use SDS-PAGE and Western-blot to identify the expression and solubility of the protein. The results are shown in Figure 3. According to the results in the figure, there are soluble expressions of chaperone protein and VP2 protein in the expression strain.

Example 2 Preparation of porcine parvovirus virus-like particles

2.1 Purification of VP2 protein and determination of VLP

2.1.1 Purification of recombinant VP2 protein

The supernatant of ultrasonic lysis was purified by Ni-NTA chromatography column (Merck). The specific process was: after Ni-NTA was equilibrated with buffer A, the sample was slowly flowed through the chromatography column, and then at least 10 times the column volume Buffer B (50mmol/L PB, 250mmol/L NaCl, 30mmol/L imidazole, pH7.0) was passed through the column to wash the impurities, and finally buffer C (50mmol/L PB, 250mmol/L NaCl, 300mmol/L imidazole , pH 7.0) to elute the target protein, collect the eluted fractions, and detect them by SDS-PAGE and Western-blot. Response was good.

2.1.2 Protein assembly and detection in vitro

Put the purified protein into a dialysis bag and perform dialysis at 4°C. By changing the salt ion concentration of the dialysate to explore the optimal conditions for VP2 protein assembly in vitro, the salt ion concentrations are set as: 50mmol/L, 100mmol /L, 150mmol/L, 200mmol/L. Then, the shape of the formed VLP was observed through a transmission electron microscope, the particle size distribution of the VLP was detected through a nanometer particle size analyzer, and the hemagglutination activity of the VLP was detected through a hemagglutination experiment. The results are shown in Figure 5. According to the results, it can be seen that the prokaryotic expressed VP2 can be assembled into VLP in vitro, and the VLP is similar to the natural PPV virus in terms of shape, size and hemagglutination activity. Further, the quality of the VLP formed at 50mmol/L NaCl, pH7.0 is better, and the formation rate is higher.

The specific detection method is as follows:

Transmission electron microscope (TEM) detection: The PPV VLP obtained from the above purification was negatively stained with 2% phosphotungstic acid, and the shape of the VLP was observed with a transmission electron microscope. The diameter of the VLP was about 24-25nm, the size was uniform, and it appeared as a hollow shape. The results are as follows Figure 6 shows.

Dynamic light scattering (DLS) detection: The PPV VLP obtained by the above purification was detected by a nanometer particle size analyzer, and the result is shown in Figure 7. According to the DLS result, the VLP obtained in the present invention has high purity, uniform size and complete structure.

Hemagglutination activity (HA) detection: Take a 96-well V-type micro-reaction plate, add 50 μL PBS to each well, add 50 μL antigen to each well in the first column, and then perform 2-fold serial dilution of the antigen until 50 μL is discarded in the 11th well. Then 50 μL of 0.8% mouse erythrocyte suspension was added to each well, mixed evenly with a micro shaker, and left at room temperature for 1 h. When judging the results, the highest dilution factor of the antigen with 100% erythrocyte agglutination was used as the judging end point.

Example 3 PPV virus-like particle immunogenicity detection

3.1 Determination of VP2 protein concentration and determination of endotoxin content

The concentration of the purified VP2 protein was determined with the BCA protein content assay kit (Pierce Biotechnology), and the specific steps refer to its instructions. The measured concentration of VP2 protein is 0.57mg/mL, and the expression level is relatively high, which can meet the requirements of industrial production.

The endotoxin content of the purified protein was determined with an endotoxin content determination kit (ToxinSensor ^TM Endotoxin Detection System, GenScript). For specific steps, refer to its instruction manual. The measured endotoxin content of the VP2 protein was less than 0.3mU/mg.

3.2 Animal Immunization

Referring to the method in "Pharmacopoeia of the People's Republic of China" (2005 edition), the VLP obtained in Example 2 was prepared into a vaccine.

3.2.1 Immunization of mice

With the participation of Freund's adjuvant, the virus-like particles were divided into 5 μg/10 μg/mouse and immunized Kunming mice aged 3-4 weeks. At the same time, the porcine parvovirus inactivated vaccine immunization group and the phosphate buffer inoculation group were set as reference As a control, 5 mice were immunized in each group. A total of 2 immunizations were performed, with Freund's complete adjuvant for the first time and incomplete Freund's adjuvant for the second time. After the first immunization, blood was collected from the tail vein every week, and the serum was separated and stored for subsequent detection of blood in mouse serum. The results showed that the virus-like particles formed by expressing the protein could stimulate the mice to produce porcine parvovirus-specific antibodies, and the level of antibodies produced was significantly higher than that of the inactivated vaccine immunized group.

3.2.2 Immunization of guinea pigs

24 female healthy guinea pigs weighing 400-500g were randomly divided into 4 groups, 6 in each group, group A and group B were immunized with oil adjuvant, 50μg/guinea pig and 25μg/guinea pig respectively, and group C was immunized with PPV inactivated vaccine As a positive control, group D was immunized with PBS as a negative control. The immunization was carried out twice, with an interval of two weeks. Heart blood was collected every 14 days after the first immunization, serum was separated to detect serum antibodies, and the spleen was aseptically removed after 64 days of death (2 rats in each group), and spleen lymphocytes were separated Detect the proliferation and secretion of cytokines in PBMC, then subcutaneously inject PPV standard poison, kill the virus 7 days after the challenge, collect tissues such as heart, liver, spleen, lung and kidney, and detect the tissue toxin content by RT-PCR. The results show that the formed virus-like particles can induce good humoral and cellular immune responses in guinea pigs, and can effectively protect the body from the virulent attack of porcine parvovirus.

3.3 Detection of antibody level

3.3.1 Detection of hemagglutination inhibitory antibodies

Preparation of 0.8% mouse red blood cells:

Use a sterilized syringe to draw 1 mL of fresh mouse blood into a sterilized centrifuge tube containing 1000 U of heparin; add 9 mL of PBS to the above centrifuge tube and centrifuge at 1500 rpm for 5 min, discard the supernatant; resuspend blood cells in 10 mL of PBS, and centrifuge at 2000 rpm After 10 minutes, the supernatant was discarded, and the erythrocytes were washed three times in this way; finally, according to the required amount, a 0.8% (v/v) mouse erythrocyte suspension was prepared with PBS and used within 5 days.

HA determines 4 units of poison price:

Carry out on a 96-well microcoagulant plate, add 50µL PBS to each well from left to right; add 50µL virus solution to the first well on the left, mix well, suck 50µL to the second well, and sequentially dilute to the 10th well Aspirate and discard 50 µL of each well; the 11th and 12th wells are red blood cell controls; after the dilution is completed, add 50 µL of 0.8% mouse red blood cell suspension to each well in turn from right to left, shake and mix on a shaker, and observe the results after standing at room temperature for 1 hour , taking the highest dilution of the virus with 100% agglutination of red blood cells as the end point, 4 times the dilution is 4 units of virus.

HI detects antibodies in serum:

Add 25 µL of PBS buffer solution to each well of a standard 96-well V-type reaction plate, test different samples in each row of wells, add 25 µL of serum to be tested in the first well of each row, mix well, take out 25 µL and add to the second well, Doubly dilute to the 10th well in turn, discard 25 µL, add positive serum to the 11th well as a positive control, and add negative serum to the 12th well as a negative control; then add 25 µL of 4 units of poison to each well, shake and mix, and incubate at room temperature for 30 minutes; Afterwards, 50 µL of 0.8% mouse erythrocyte suspension was added to each well, incubated at room temperature for 2 hours to observe the results, and the highest dilution of serum that completely inhibited erythrocyte agglutination was used as the hemagglutination inhibitory titer of the serum. As shown in Figure 8, the hemagglutination inhibition titer was 1:5120.

3.3.2 Neutralizing antibody detection

Porcine parvovirus standard strain virus titer determination:

The virus species used in this test method is the standard strain of porcine parvovirus, which was purchased from the China Veterinary Drug Control Institute. This strain can proliferate on PK-15 cells and has good immunogenicity to pigs. Determination of virus content: PK-15 cells were inoculated in 96-well cell culture plates at 2×10 ⁵ /mL. When the cells covered 80% of the bottom of the wells, they could be used for virus inoculation; the virus seeds were continuously inoculated 10 times with DMEM culture medium. For serial dilution, 100 µL of each dilution was added to the above-mentioned 96 _- well cell culture plate, and each dilution was repeated 8 times, and a normal cell culture control was set. Discard the virus solution, add 100 µL of DMEM maintenance solution containing 3% FBS to each well, and place in a 37°C, 5% CO2 incubator to continue culturing; observe cell changes day by day, cell pyknosis, aggregation, increased granules, and some cell fusion , shedding, and more voids are judged as CPE. After 3 days of observation, the number of wells with cytopathic changes was recorded, and the TCID ₅₀ of the virus was calculated according to the Reed-Muench method. Results: The virus liquid per milliliter was not less than 10 ^6.0 TCID ₅₀ .

Serum neutralizing antibody detection:

The detection of porcine parvovirus neutralizing antibody is carried out with reference to the standard in "Pharmacopoeia of the People's Republic of China" (2005 edition), and the specific implementation method is: the sample serum is inactivated at 56°C for 30min, and then in a 96-well cell culture plate at a ratio of 1:10 The initial concentration was diluted to the 10th well, then 100 TCID ₅₀ of 7909 strains of porcine parvovirus was added to each well, mixed evenly, placed in a 37°C, 5% CO2 incubator and incubated for 2 hours, and then 100µL was inoculated in the previously prepared In a 96-well cell culture plate with 80% PK-15 cells, place it in an incubator containing 5% CO2 at 37°C for 2 days, then observe the cell lesion, and take the highest dilution of serum at which 50% of the cells are lesioned as the Serum neutralizing antibody titers. The results showed that the serum neutralizing antibody titer was about 1:420.

SEQUENCE LISTING

<110> Henan Academy of Agricultural Sciences

<120> A method for preparing porcine parvovirus virus-like particle subunit vaccine using Escherichia coli expression system and

application

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Claims (9)

1. encoding a gene for PPV VP 2 protein, its sequence is as shown in SEQ ID NO.1.

2. an expression vector, containing the gene order described in claim 1.

3. a host cell, containing the expression vector described in claim 2, and can be with the companion of this expression vector coexpression Protein expression vector.

4. host cell as claimed in claim 3, it is characterised in that: described PPV VP 2 protein is solubility expression.

5. the solubility expression method of a PPV VP 2 protein, it is characterised in that: by PPV VP 2 protein with Chaperone co expression.

6. the solubility expression method of as claimed in claim 5 PPV VP 2 protein, it is characterised in that specifically include with Lower step:

A. design and synthesize codon optimized pig parvoviral VP2 gene, be attached with pET28a carrier, build protokaryon table Reach carrier；

B. VP2, the E. coli recombinant stain of chaperone coexpression are built；

C. recombinant bacterial strain inoculation while add tetracycline, 37 DEG C cultivate 2～3h, to OD value reach 0.6 time, addition The expression of 1.0mmol/L isopropylthiogalactoside induction VP2 albumen.

7. the expression of a pig parvoviral virus sample granule, it is characterised in that comprise the following steps:

By claim 6 gained inducible strain ultrasonic degradation, the supernatant after cracking passes through affinitive layer purification, by the product of purification Thing buffer is dialysed, and to obtain final product.

8. the expression of pig parvoviral virus sample granule as claimed in claim 7, it is characterised in that: the ring of described dialysis Border condition is: 4 DEG C, 50mmol/L NaCl, pH7.0.

9. the application in preparation subunit vaccine of the pig parvoviral virus sample granule expressed by claim 7.