CN116102660A

CN116102660A - Porcine parvovirus gene engineering epitope vaccine and preparation method thereof

Info

Publication number: CN116102660A
Application number: CN202211138719.2A
Authority: CN
Inventors: 李群; 仇义; 丁国伟; 李甜甜; 魏荣荣; 李琛; 徐萍; 陈林中日; 荣雪路; 潘晨; 王设市; 陈森
Original assignee: Yangzhou Uni Bio Pharmaceutical Co ltd
Current assignee: Yangzhou Uni Bio Pharmaceutical Co ltd
Priority date: 2022-09-19
Filing date: 2022-09-19
Publication date: 2023-05-12
Anticipated expiration: 2042-09-19
Also published as: CN116102660B

Abstract

The invention discloses a porcine parvovirus gene engineering epitope vaccine and a preparation method thereof, belonging to the field of biological products for animals. The preparation method of the vaccine provided by the invention is that hepatitis B virus core antigen protein is taken as a carrier, fusion epitopes constructed by porcine parvovirus VP2 protein Th epitopes, CTL epitopes and B cell epitopes are inserted between 79 Th amino acid and 80 Th amino acid of hepatitis B virus core protein (HBc) genes, and insect cell-baculovirus expression systems are utilized to construct and express, and the recombinant virus efficiently expresses antigen protein in insect cell HF; extracting, purifying and BEI inactivating, adding adjuvant, and emulsifying to obtain vaccine. The preparation method is simple, can prepare a large amount of antigen proteins, has short time consumption and high expression quantity, greatly reduces the production cost and is beneficial to large-scale production. The porcine epidemic diarrhea subunit vaccine containing the HBcAg-PPV-Epitops recombinant protein prepared by the invention has good immune effect and small immune dose, and can effectively prevent porcine parvovirus infection.

Description

Porcine parvovirus gene engineering epitope vaccine and preparation method thereof

Technical Field

The invention relates to a porcine parvovirus gene engineering epitope vaccine and a preparation method thereof, belonging to the field of biological products for animals.

Background

Since no effective medicine or treatment method for PPV exists at present, the prevention and immunization of the disease are particularly important. In recent years, the use of vaccines has been acknowledged as the only method for preventing porcine parvovirus infection and improving sow reproductive rate. PPV vaccines, attenuated live vaccines, inactivated vaccines, genetically engineered subunit vaccines, genetically engineered live viral vector vaccines, genetic vaccines and the like are developed in more than 10 countries. The attenuated vaccine is a complete pathogenic vaccine which can weaken pathogenic pathogenicity and still has activity through artificial weakening or natural screening, and can induce the comprehensive immune response of organisms. The earliest found and clinically used is the NADL-2 attenuated strain, which is attenuated by PPV after more than 50 passages in the laboratory using cell culture. The strain can produce good immunity effect by oral administration or nasal drip, and can not infect fetus through placenta. Currently, most live vaccines used in the market are attenuated vaccines. Although a plurality of attenuated vaccines are clinically applied, the concern of people about virus recombination and attenuated virulence return is always that the application of the attenuated vaccine is limited due to the existence of a large number of virulent strains. The genetic engineering live virus vector vaccine is constructed by inserting protective antigen genes and promoter sequences of heterologous viruses into another virus serving as a vector by utilizing genetic engineering technology, and can induce specific immune response aiming at the vector viruses and the heterologous viruses in an organism. The nucleic acid vaccine is also called gene vaccine, which is to directly inject DNA or RNA encoding target protein gene and expression control sequence into animal organism, and synthesize target protein by using transcription and translation system of organism to stimulate organism to produce specific humoral immunity and cell immunity response. Such nucleic acids capable of expressing foreign proteins in vivo and stimulating an immune response in the body are known as nucleic acid vaccines, which include DNA and RNA vaccines. The nucleic acid vaccine is simple and convenient to produce, has the characteristics of safety of an inactivated vaccine, full immune response induction of a attenuated vaccine and the like, but whether the gene expression vector is integrated with a host chromosome or not needs further research.

The core antigen (HBcAg) of Hepatitis B Virus (HBV) has special structural characteristics, and the total length of the antigen is 183 amino acids, so that the antigen can be assembled into particles with a size of two. It has been found that amino acid residues 78-83 are located at the tip of spike, insertion of foreign fragment at the site of spike will not affect granule formation, and the product can be folded and assembled correctly in cells, and can be expressed in both eukaryotic and prokaryotic cells with high efficiency.

Therefore, the development of the production method of the porcine parvovirus subunit vaccine with low production cost, high production efficiency and good vaccine immunity effect has important practical significance.

Disclosure of Invention

The invention aims to provide a recombinant protein expressed by a baculovirus-insect cell expression system by taking a human hepatitis B core antigen as a vector and inserting fusion genes of a plurality of epitopes of porcine parvovirus VP2 protein between 79-80 amino acids, wherein the recombinant protein can be self-assembled into virus-like particles (Virus Like Particles, VLPs) in vitro, and main epitopes of the PPVVP2 protein are displayed on the surface of the VLP. The genetic engineering vaccine prepared by the recombinant protein has the advantages of high efficiency, good safety, high antibody uniformity and high protection rate, thereby overcoming the defects of the prior art.

A first object of the present invention is to provide a recombinant antigen protein obtained by passing the PPV-Epitops gene through a flexible linker (GGGGS) ₅ The connection mode is inserted between 79-80 amino acid sequences of HBcAg molecules of the hepatitis B virus.

In one embodiment, the amino acid sequence of the HBcAg molecule of the hepatitis B virus is shown as SEQ ID NO.1, and the amino acid sequence of the PPV-Epitops gene is shown as SEQ ID NO. 3.

In one embodiment, the nucleotide sequence of the HBcAg molecule of the hepatitis B virus is shown as SEQ ID NO.2, and the nucleotide sequence of the PPV-Epitops gene is shown as SEQ ID NO. 4.

In one embodiment, the amino acid sequence of the recombinant antigen protein is shown in SEQ ID NO.5.

In one embodiment, the nucleotide sequence of the recombinant antigen protein is shown in SEQ ID NO. 6.

A second object of the present invention is to provide a gene encoding the above recombinant antigen protein, the nucleotide sequence of which is shown as SEQ ID NO. 2.

A third object of the present invention is to provide a genetically engineered epitope vaccine for preventing porcine parvovirus, which contains the recombinant antigen protein.

In one embodiment, the amino acid sequence of the recombinant antigen protein is as shown in SEQ ID NO.5.

In one embodiment, the recombinant antigenic protein is subjected to BEI inactivation.

In one embodiment, the recombinant antigen protein is present in an amount of 50 to 100 μg/mL.

In one embodiment, the genetically engineered vaccine further comprises an adjuvant.

In one embodiment, the adjuvant includes, but is not limited to: aluminum salt adjuvants, liposome adjuvants, nanoadjuvants, and saponin adjuvants.

In one embodiment, the adjuvant is a nanoadjuvant.

The invention also provides application of the recombinant antigen protein or the genetically engineered epitope vaccine in preparing medicines for preventing and/or treating porcine parvovirus.

The beneficial effects are that:

the recombinant baculovirus rBac-HBcAg-PPV-Epitopes of the expressed recombinant protein is connected into insect cells to efficiently express HBcAg-PPV-Epitopes protein, cell fragments are removed through centrifugation, BEI is added for inactivation, and then nano-adjuvant is added for mixing and emulsification to prepare the vaccine. The vaccine prepared by the invention can improve the antibody level after immunization, improve the uniformity of the antibody after immunization, ensure the immune effect of the vaccine, and has the advantages of high efficiency and good safety.

Drawings

FIG. 1 is a schematic representation of a fused HBcAg molecule carrying a foreign gene;

FIG. 2 is a transmission electron micrograph of the recombinant protein after purification.

Detailed Description

Example 1: construction of fusion Gene fragment HBcAg-PPV-Epitopes

1. Construction of fusion gene segment HBcAg-PPV-Epitopes: as shown in FIG. 1, fusion epitope PPV-Epitops genes constructed by porcine parvovirus VP2 protein Th Epitopes, CTL Epitopes and B cell Epitopes are inserted between 79-80 amino acid sequences of HBcAg molecules by using molecular biological technology and method, and fusion gene fragments HBcAg-PPV-Epitops are constructed (the nucleotide sequence is shown as SEQ ID NO. 6).

2. The target gene is connected with the transit plasmid: the expression vectors pFastBac I and HBcAg-PPV-Epitops gene fragments were digested with BamH I and HindI, recovered and purified, and ligated overnight at 4℃with T4 DNA ligase to obtain ligation products.

3. Ligation product transformation competent cells: and (3) transforming the ligation product obtained in the step (2) into T1 competent cells under the aseptic condition, uniformly mixing, carrying out ice bath for 30min, carrying out heat shock for 90s at 42 ℃, immediately carrying out ice bath for 2min, and adding 800 mu L of LB culture under the aseptic condition for 60min based on shaking culture at 37 ℃. The culture was centrifuged at 14000rpm for 1min, 800. Mu.L of the supernatant was aspirated, and the remaining culture was plated on LB (ampicillin-resistant) solid medium and cultured overnight at 37 ℃. Single colony is selected for colony PCR identification, and positive plasmid is detected and sequenced. The recombinant plasmid with correct sequencing was named pFastBac I-HBcAg-PPV-Epitopes.

4. Recombinant baculovirus construction: transferring the recombinant plasmid pFastBac I-HBcAg-PPV-Epitopes in the step 3 into competent cells of escherichia coli DH10 Bac, and selecting positive clones to use M13 primers for PCR identification.

M13-F：TGTAAAACGACGGCCAGT

M13-R：CAGGAAACAGCTATGAC

The PCR reaction system was (total volume 25. Mu.L): DNA template 0.5 u L, M-F and M13-R0.5 u L, DNA polymerase 12.5 u L and sterile water 11 u L. The PCR reaction conditions were: 95 ℃ for 5min;95 ℃ for 30s,65 ℃ for 30s,72 ℃ for 90s,30 cycles; and at 72℃for 10min.

The PCR products were subjected to 1% agarose gel electrophoresis, and the results showed that a specific band of about 3000bp was successfully amplified, consistent with the expected size. Positive recombinant bacmid was named rBacmid-HBcAg-PPV-Epitopes.

5. Recombinant bacmid transfection sf9 cells: and (3) transfecting the recombinant bacmid-HBcAg-PPV-Epitops purified in the step (4) into sf9 cells by using a liposome transfection method, wherein the specific operation method is carried out by referring to a cellfectin transfection reagent instruction book of Semer Feier technology (China) limited company, and the f1 generation recombinant baculovirus rBac-HBcAg-PPV-Epitops is obtained.

Example 2: preparation of recombinant HBcAg-PPV-Epitops protein

1. Amplification of recombinant baculovirus: inoculating the f 1-generation recombinant baculovirus rBac-HBcAg-PPV-Epitops in the example 1 with insect cells sf9, culturing for 4 days at 27 ℃, collecting the culture, centrifuging and taking the supernatant to obtain the f 2-generation recombinant baculovirus;

2. identification of expressed protein:

(1) Inoculating the f2 generation recombinant baculovirus into insect cells sf9 according to the inoculum size of MOI=5-10, culturing for 4 days at 27 ℃, collecting a culture, and centrifuging to obtain a supernatant to obtain recombinant HBcAg-PPV-Epipops protein;

(2) SDS-PAGE identification: subjecting the supernatant to SDS-PAGE electrophoresis; after electrophoresis, the molecular weight of the sample at about 30kDa was found to match the theoretical size after staining and decolorizing, indicating successful expression.

(2) Western Blot identification: and (3) taking gel after SDS-PAGE electrophoresis, directly transferring the gel to an NC film by using a BIO-LAB transfer device, and performing Western blot identification according to a conventional method after transfer. Porcine epidemic diarrhea positive serum reference (1:200) was used as primary antibody (positive serum after PPV challenge); goat anti-pig IgG marked by horseradish peroxidase (1:2000) is used as an enzyme-labeled secondary antibody; finally, color development was performed with TMB (Biyun Tian Biotechnology institute). The result shows that 1 obvious specific band appears at 30kDa, and the negative control has no specific reaction, so that the recombinant protein can be recognized by antibodies in porcine parvovirus positive serum, and has good specificity and reactivities.

3. High expression of recombinant HBcAg-PPV-Epitops protein: inoculating the identified correct recombinant virus into HF cells in a virus-receiving amount with MOI=1-10 for mass culture, and centrifugally collecting culture solution supernatant to obtain recombinant HBcAg-PPV-Epitops protein with mass.

4. Purifying recombinant HBcAg-PPV-Epitops protein: the recombinant protein was purified using a GE nickel column (HisTrap HP,5 mL) and the purified recombinant protein was dialyzed to remove imidazole and sodium chloride from the purified sample.

5. Quantification of recombinant HBcAg-PPV-Epitops protein: protein quantification was performed using the BCA protein concentration assay kit (enhanced type) from Biyun institute biotechnology, and the result showed that the concentration of the purified target protein was 2.03mg/mL, and the transmission electron microscope results were shown in FIG. 2.

Example 3 preparation of porcine parvovirus genetically engineered subunit vaccine

The purified HBcAg-PPV-Epitops recombinant protein obtained in example 2 was taken, emulsified by adding an adjuvant, mixed well and stored at 4 ℃. The specific proportions of the vaccine are shown in Table 1.

TABLE 1 pig parvovirus genetic engineering subunit vaccine composition ratio

Example 4 immunogenicity test of porcine parvovirus genetically engineered vaccine

1 replacement gilt immunopotentiation test

1.1 immunization 3 batches of vaccine 1, vaccine 2 and vaccine 3 prepared in laboratory are immunized with 1 ml/head respectively to prepare a 5-6 month-old backup sow, 4 heads/group, 3 groups are added, and the neck intramuscular injection is carried out. Two additional sows were taken as control groups, each injected with the same dose of sterile PBS. Blood was collected 4 weeks and 9 weeks after the immunization, and serum antibodies were detected by a trace amount of blood coagulation inhibition test.

1.2 breeding sow healthy boars (porcine parvovirus antigen, antibody were negative) were selected for breeding one month after immunization.

1.3 challenge pregnancy about 40 days, taking 3 groups of immunized replacement gilts and 2 control gilts for challenge, and diluting the virulent BJ-2 strain F3 virus seed for test until the virus content is 10 ^5.0 TCID ₅₀ And (3) carrying out nasal drip inoculation, carrying out 4 ml/head isolation feeding, observing delivery every day, and recording whether the phenomena of small abdominal circumference, abortion, stillbirth, mummy and the like occur.

1.4 detection of viremia of sows and piglets after virus challenge, blood collection is carried out on pregnant sows 5 days, 7 days and 9 days after virus challenge, blood collection is carried out on piglets with healthy eyes after delivery, plasma is separated, the piglets are inoculated to well-grown ST monolayer cells after aseptic treatment, the piglets are cultured for 5 days at 37 ℃, and PCR identification is carried out by using porcine parvovirus specific primers. The primer sequences were as follows:

PPV-P1：5’-TGGTCTCCTTCTGTGGTAGG-3’

PPV-P2：5’-CAGAATCAGCAACCTCAC-3’

the conditions should be as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30 seconds, annealing at 65℃for 1 minute, extension at 72℃for 30 seconds, total of 35 cycles; finally, the extension was carried out at 72℃for 7 minutes.

The PCR products were identified by 1% agarose gel electrophoresis.

1.5 guinea pig immunopotentiation test

1.5.1 immunization 3 batches of vaccine 1, vaccine 2, vaccine 3 were immunized with 5 healthy guinea pigs, 0.25 ml/3, 3 total groups, i.m. leg, and 5 control groups, respectively, were injected with the same dose of sterile PBS.

1.5.2 serum HI antibody assay groups were collected at 4 weeks post-inoculation, serum was isolated and PPV HI antibody titers were determined by microagglutination.

2 results

2.1 antibody detection results after immunization of the backup sow are shown in Table 2, the serum HI antibodies of the immunized groups of the vaccine of different batches are not lower than 1:64 in 4 weeks after immunization of each group, and the serum antibodies are increased by 1-2 titer in 9 weeks after immunization; the serum HI antibody of the control group is not higher than 1:8.

TABLE 2 detection results of antibodies after immunization of different batches of vaccine (Nlog 2)

2.2 the results of the challenge of pregnant sows are shown in Table 3, and the results show that after the pregnant sows of the immune group are subjected to the challenge with BJ-2 strong strain, all vaccine immune groups are 100% protected after the challenge, and no abortion, dead fetus, mummy fetus and piglet viremia exists; control group 2/2 produced dead and mummy fetuses, and other surviving piglets detected viremia.

TABLE 3 results of toxicity test for pregnant sow

2.3 results of guinea pig immunopotency

The HI titers of 3 batches of vaccine are not lower than 1:64 after 4 weeks of guinea pig immunization, and the geometric average values are 6.8log2, 6.6log2 and 6.4log2 respectively. The serum antibodies of the blank control group are all negative. See table 4.

Table 4 serum HI antibody levels (Nlog 2) from 4 weeks immunized guinea pigs with different vaccine batches

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A recombinant antigen protein is characterized in that the PPV-Epitops gene is passed through a flexible linker (GGGGS) ₅ The connection mode is inserted between 79-80 amino acid sequences of HBcAg molecules of the hepatitis B virus.

2. The recombinant antigen protein according to claim 1, wherein the amino acid sequence of the hepatitis b virus HBcAg molecule is shown in SEQ ID No.1, and the amino acid sequence of the PPV-ephops gene is shown in SEQ ID No. 3.

3. A gene encoding the recombinant antigen protein of claim 1 or 2, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 6.

4. An epitope vaccine for preventing porcine parvovirus gene engineering, characterized by comprising the recombinant antigen protein of claim 1 or 2.

5. The genetically engineered epitope vaccine of claim 4, wherein said recombinant antigenic protein is BEI inactivated.

6. The genetically engineered epitope vaccine of claim 4, wherein the recombinant antigenic protein is present in the genetically engineered vaccine in an amount of 50-100 μg/mL.

7. The genetically engineered epitope vaccine of claim 4, further comprising an adjuvant.

8. The genetically engineered epitope vaccine of claim 7, wherein said adjuvant is selected from the group consisting of an aluminum salt adjuvant, a liposome adjuvant, a nanoadjuvant, and a saponin adjuvant.

9. The genetically engineered epitope vaccine of claim 8, wherein said adjuvant is a nanoadjuvant.

10. Use of the recombinant antigenic protein of claim 1 or the genetically engineered epitope vaccine of any one of claims 4 to 9 in the preparation of a medicament for preventing and/or treating porcine parvovirus.