CN112725370B

CN112725370B - PRRSV ORF5 fusion gene DNA vaccine and preparation method thereof

Info

Publication number: CN112725370B
Application number: CN202011609207.0A
Authority: CN
Inventors: 刘建奎; 魏春华; 林志锋; 徐叶
Original assignee: Longyan University
Current assignee: Longyan University
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-09-06
Anticipated expiration: 2040-12-30
Also published as: CN112725370A

Abstract

The invention discloses a PRRSV ORF5 fusion gene DNA vaccine and a preparation method thereof. Belongs to the field of biotechnology. The invention utilizes the DNA shuffling technology to carry out in-vitro mutation recombination on PRRSV ORF5 genes of 4 lineages, and two recombinant plasmids PCA-delta ORF5-8 and PCA-delta ORF5-46 are screened, so that a neutralizing antibody can be generated more effectively, and a foundation is laid for further research and development of safe and effective PRRSV DNA vaccines.

Description

PRRSV ORF5 fusion gene DNA vaccine and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a PRRSV ORF5 fusion gene DNA vaccine and a preparation method thereof.

Background

Porcine Reproductive and Respiratory Syndrome (PRRS) is a highly contagious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), has spread to most swine-breeding countries, becomes a global viral infectious disease of pigs, and causes huge economic losses in all countries of the world. At present, based on the Type 2PRRSV classification system, the epidemic strains in China are divided into four pedigrees: pedigree 1(NADC30-like), pedigree 3 (QYYYZ-like), pedigree 5 subline 5.1(VR2332-like) and pedigree 8 subline 8.7(JXA1-like and CH-1a), and the strain sequences of the various pedigrees have large differences, wherein HP-PRRSV in the pedigree 8 subline 8.7 and the NADC30-like strains in the pedigree 1 are the dominant strains of the epidemic strains in China. Particularly, the similar NADC30PRRSV which appears in 2012 domestically has high genome variation among strains, is easy to recombine with other strains including vaccine strains, so that the commercialized vaccine cannot provide good immune protection for the strains, and brings serious challenges to the pig industry in China.

The study shows that GP5 encoded by ORF5 is the most main protective antigen of PRRSV and can induce organism to generate neutralizing antibody. In addition, after the PRRSV invades the organism, a neutralizing antibody aiming at GP5 protein is mainly generated, so that GP5 protein becomes the first choice protein for developing a novel vaccine.

In conclusion, how to provide a PRRSV vaccine is a problem that needs to be solved urgently by those skilled in the art.

Disclosure of Invention

In view of the above, the invention utilizes the DNA shuffling technology to carry out in vitro mutation recombination on PRRSV ORF5 genes of 4 lineages, screens two recombinant plasmids PCA-delta ORF5-8 and PCA-delta ORF5-46, and prepares the porcine reproductive and respiratory syndrome vaccine by using the two recombinant plasmids.

In order to achieve the purpose, the invention adopts the following technical scheme:

a PRRSV ORF5 fusion gene, which contains partial ORF5 genes of 4 lineages at the same time;

the 4 lineages are lineage 1, lineage 3, lineage 5 subline 5.1, and lineage 8 subline 8.7.

Further, the nucleotide sequence is as follows:

ΔORF5-8：AACAGCAGCTCAAATTTACAGTTGATTTATAACCTGACGATATGTGAGCTGAATGGCACAGACTGGCTGGACAAAAAATTTGATTGGGCAGTGGAGACTTTCGTCATCTTCCCTGTATTGACTCACATAGTCTCTTACGGCGCCCTCACCACTAGTCATTTTCTTGACACGGTCGGCCTGGCCACTGTGTCTACCGCCGGTTATTATCATGGGCGGTATGTGTTGAGTAGTATTTACGCTGTCTGTGCCCTGGCTGCGTTGGTTTGCTTCGCCATTAGGTTGGCGAAAAATTGCATGTCCTGGCGTTACTCATGCACCAGATATACTAATTTTCTCCTGGATACTAAGGGCACACTCTACCGCTGGCGGTCACCCGTCATCATTGAGAAAGGGGGTAAAGTTGATGTCGGGGGTCATTTAATCGACCTCAAAAGAGTTGTGCTTGATGGTTCCGCGGCTACCCCT；SEQ ID No.1；

or Δ ORF 5-46: AACAGCAGCTCAAATTTACAGTTGATCTATAACCTGACGATATGTGAGCTGAATGGCACAGATTGGCTGGCACAAAAATTTGACTGGGCGGTGGAGATTTTTGTCATCTTCCCCGTGTTGACCCACATTGTTTCCTATGGGGCACTCACCACCAGCCATTTCCTTGACACAGTTGGTCTGGCCACTGTGTCCACTGCCGGATATTATCACGGGCGGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCTCTGGCAGCGCTGATTTGCTTTGTCATTAGGCTTGCGAAGAACTGCATGTCCTGGCGCTACTCTTGTACCAGATATACCAACTTTCTTCTGGACACTAAGGGCAAAATCTATCGTTGGCGGTCACCCGTCATCATAGAGAAAGAGGGTAAAGTTGAGGTCGGTGGCCATCTCATTGACCTCAGGAGAGTCGTGCTTGATGGTTCCGCGGCTACCCCT, respectively; SEQ ID No. 2.

Further, the amino acid sequence encoded by Δ ORF5-8 is as follows:

NSSSNLQLIYNLTICELNGTDWLDKKFDWAVETFVIFPVLTHIVSYGALTTSHFLDTVGLATVSTAGYYHGRYVLSSIYAVCALAALVCFAIRLAKNCMSWRYSCTRYTNFLLDTKGTLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATP；SEQ ID No.3；

the amino acid sequence encoded by Δ ORF5-46 is as follows:

NSSSNLQLIYNLTICELNGTDWLAQKFDWAVEIFVIFPVLTHIVSYGALTTSHFLDTVGLATVSTAGYYHGRYVLSSIYAVCALAALICFVIRLAKNCMSWRYSCTRYTNFLLDTKGKIYRWRSPVIIEKEGKVEVGGHLIDLRRVVLDGSAATP；SEQ ID No.4。

a porcine reproductive and respiratory syndrome vaccine containing the fusion gene.

Further, a eukaryotic expression vector, PCAGGS-HA, was used for construction.

According to the technical scheme, compared with the prior art, the invention has the following beneficial effects: the DNA shuffling technology can enable the gene to be recombined at a molecular level, thereby screening out a mutant which has expected multiple parent genes. The invention utilizes the DNA shuffling technology to carry out in vitro mutation recombination on PRRSV ORF5 genes of 4 lineages and screen two recombinant plasmids PCA-delta ORF5-8 and PCA-delta ORF5-46, thereby being capable of more effectively generating neutralizing antibodies and laying a foundation for further developing safe and effective PRRSV DNA vaccines.

Drawings

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

FIG. 1 is a graph showing the results of indirect immunofluorescence assay of protein expression in MARC-145 cells using recombinant plasmids according to example 3 of the present invention;

FIG. 2 is a drawing showing the results of the WesternBlot assay for protein expression in MARC-145 cells by recombinant plasmids in example 3 of the present invention, wherein 1 represents. DELTA. ORF5-8, and 2 represents. DELTA. ORF 5-46;

FIG. 3 is the result of GP5 protein detection in the serum of mouse in example 4 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental materials required in the examples of the present invention are as follows:

the PCAGGS-HA (PCA-HA) vector plasmid is purchased from Hunan Fenghui Biotech Co., Ltd;

mouse anti-HA protein antibodies were purchased from Beijing-Qiao Shenzhou technologies, Inc.;

the Lp8000 transfection kit and the ECL hypersensitive luminescence kit are purchased from Biyuntian biotechnology limited company;

the PCA-ORF5-MLV (ORF5 gene from vaccine) plasmid is provided by livestock infectious disease prevention and treatment and biotechnology key laboratory in Fujian province;

the endotoxin-free large quality-improving particle kit is purchased from OMEGA company;

the mouse interleukin-4 and interferon-gamma ELISA kit is purchased from West pool science and technology limited company;

mouse PRRSV GP5 protein (PRRSV-GP5) ELISA kits were purchased from shanghai luyu biotechnology limited;

cell Counting kit (cck8) was purchased from Bilun sky Biotechnology, Inc.

Other biochemical reagents are all domestic analytical pure products.

8 PRRSV FJ-Chen, FJ-Huang and FJ-Yang (lineage 1), FJ-Deng and FJ-Liu (lineage 3 strain), FJ-Wang (lineage 5 subline 5.1 strain), FJ-Qiu and FJ-Fu (lineage 8 subline 8.7 strain) of different lineages, and Marc-145 cells used in the study were provided by livestock infectious disease control and biotechnology focus laboratories of Fujian province.

PMD-19T vector was purchased from Takara, PRRSV N protein monoclonal antibody (MAb) from Xiamen Ruibetai Biomedicine, Inc., FITC-labeled goat anti-mouse IgG from Biyunnan Biotechnology, Inc.

The unrecited experimental method is a conventional experimental method, and is not described in detail herein.

Example 1 construction of recombinant plasmid by DNA shuffling technology, screening of recombinant plasmid

(1) Viral RNAs were extracted from FJ-Chen (lineage 1), FJ-Huang (lineage 1), FJ-Yang (lineage 1), FJ-Deng (lineage 3), FJ-Liu (lineage 3), FJ-Wang (lineage 5 subline 5.1), FJ-Qiu (lineage 8 subline 8.7) and FJ-Fu (lineage 8 subline 8.7), and the ORF5 gene was amplified. The nucleotide sequence is as follows:

FJ-Chen：GACAGCAGCTCCCATTTACAGTTGATCTATAACCTGACGATATGTGAGCTGAATGGCACAGATTGGCTTGACAAGAAATTTGACTGGGCAGTGGAGACTTTCGTCATCTTCCCTGTGCTTACCCATATTGTCTCTTACGGTGCCCTCACCACCAGCCATTTTCTTGACACGGTCGGCCTGATCACTGTGTCCACCGCCGGTTATTATCATGGGCGGTATGTTTTGAGTAGCATCTACGCTGTCTGTGCCCTGGCTGCGTTTGTTTGCTTCACCATTAGGTTGGCTAAAAATTGCATGTCCTGGCGCTACTCATGCACCAGATATACTAATTTTCTTCTGGATACTAAGGGTAAACTCTATCGCTGGCGGTCATCCGTCATCATAGAGAAAGGGGGTAAAGTTGATGTTGGGGGTCATTTAATCGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCT；SEQ ID No.5；

FJ-Huang：GACAGCAGCTCCCACTTACAATTGATTTATAACCTAACGATATGTGAGCTGAATGGCACAGATTGGCTGAACAAAAAATTTGACTGGGCAGTGGAGACTTTCGTTATTTTCCCTGTGTTGACTCATATTGTCTCTTACGGCGCCCTCACCACTAGCCATTTTCTTGACACGGTTGGCCTGATCACTGTATCCACCGCCGGTTACTATCGTGGGCGGTATGTATTGAGTAGCATCTACGCTGTCTGTGCCCTGGCTGCGTTTGTTTGCTTCGCCATTAGGTTGGCAAAAAATTGTGTGTCCTGGCGCTACTCATGCACCAGATATACCAATTTTCTTCTGGATACTAAGGGCAAGCTATATCGCTGGCAGTCATCCGTCATCATAGAGAAGGGGGGCAAAGTTGATGTGGGGGGTCATTTAATCGACCTCAAGAGAGTCGTGCTTGATGGTTCTGCGGCAACCCCT；SEQ ID No.6；

FJ-Yang：AACAGCAGCTCCCACTTACAGTCGATTTATAACCTGACGATATGTGAGCTGAATGGCACAGATTGGCTGAACAAAAATTTTGACTGGGCAGTAGAGACTTTCGTTATTTTTCCTGTGCTGACTCATATTGTCTCTTACGGCGCCCTCACCACTAGCCATTTTCTTGACACAATCGGCTTGATCACTGTGTCCACCGCCGGTTATTATCACGGGCGGTATGTATTGAGTAGCATCTACGCTGTCTGTGCCCTGGCTGCGTTCGTTTGCTTCGCCATTAGGTTGGCAAAAAATTGTATGTCCTGGCGCTACTCATGCACCAGATATACCAATTTTCTTCTGGATACCAAGGGCAAACTCTATCGCTGGCGGTCACCCGTCATCATAGAGAAAGGGGGCAAAGTTGATGTTGGGGGTCATTTAATCGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCT；SEQ ID No.7；

FJ-Deng：AACAGCAGCTCTTATTCGCAGTTGATTTATAACCTAACGCTATGTGAGCTGAATGGCACAGAGTGGCTGGCCACTAGATTTGATTGGGCAGTGGAATGTTTTGTCATCTTTCCCGTGTTGACTCACATCATCTCCTACGGTGCCCTCACTACCAGCCATTTTCTTGACACAATTAGCCTGGTCACTGTATCCACCGCCGGGTATTATCACGGGCGGTATGTTTTGAGTAGTATCTACGCAGTCTGTGCTTTTGCTGCGTTAATCTGCTTTGTCATTAGGTTGGCGAAGAATTGCATGTCTTGGCGCTGCTCATGTACCAGGTATACTAATTTTCTTCTTGATACTAAGGGCAAAATCTATCGTTGGCGGTCACCCGTCATCATAGAGAAAGAGGGTAAAGTTGAGGTCGGTGGCCATCTCATTGACCTCAAGAGAGTCGTGCTTGATGGTTCCGCGGCAACCCTT；SEQ ID No.8；

FJ-Liu：AACAGCAGCTCTTATTCGCAGTTGATTTATAACCTAACGCTATGTGAGCTGAATGGCACAGAGTGGCTGGCCACTAGATTTGATTGGGCAGTGGAATGTTTTGTCATCTTTCCCGTGTTGACTCACATCATCTCCTACGGTGCCCTCACTACCAGCCATTTTCTTGACACAGTTAGCCTGGTCACTGTATCCACCGCCGGGTATTATCACGGGCGGTATGTTTTGAGTAGTATCTACGCAGTCTGTGCTTTTGCTGCGTTAATCTGCTTTGTCATTAGGTTGGCGAAGAATTGCATGTCTTGGCGCTACTCATGTACCAGGTATACTAATTTTCTTCTTGATACTAAGGGCAAAATCTATCGTTGGCGGTCACCCGTCATCATAGAGAAAGAGGGTAAAGTTGAGGTCGGTGGCCATCTCATTGACCTCAAGAGAGTCGTGCTTGATGGTTCCGCGGCAACCCTT；SEQ ID No.9；

FJ-Wang：GACAGCAGCTCCCATCTACAGCTGATTTACAACTTGACGCTATGTGAGCTGAATGGCACAGATTGGCTAGCTAACAAATTTGATTGGGCAGTGGAGAGTTTTGTCATCTTTCCCGTTTTGACTCACATTGTCTCCTATGGTGCCCTCACTACCAGCCATTTCCTTGACACAGTCGCTTTAGTCACTGTGTCTACCGCCGGGTTTGTTCACGGGCGGTATGTCCTAAGTAGCATCTACGCGGTCTGTGCCCTGGCTGCGTTGACTTGCTTCGTCATTAGGTTTGCAAAGAATTGCATGTCCTGGCGCTACGCGTGTACCAGATATACCAACTTTCTTCTGGACACTAAGGGCGGACTCTATCGTTGGCGGTCGCCTGTCATCATAGAGAAAAGGGGCAAAGTTGAGGTCGAAGGTCATCTGATCGACCTCAAAAGAGTTGTGCTTGATGGTTCCGTGGCAACCCCT；SEQ ID No.10；

FJ-Qiu：GACAACAGCTCTCATATTCAGTTGATTTATAACTTAACGTTATGTGAGCTGAATGGCACAGATTGGCTGGCACAAAAATTTGACTGGGCAGTGGAGACTTTTGTCATCTTCCCCGTGTTGACTCACATTGTTTCCTATGGGGCACTCACCACCAGCCATTTCCTTGACACAGTTGGTCTGGCCACTGTGTCCACCGCCGGATATTATCACGGGCGGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCTCTGGCTGCGCTGATTTGCTTTGTCATTAGGCTTGCGAAGAACTGCATGTCCTGGCGCTACTCTTGTACCAGATATACCAACTTCCTTCTGGACACTAAGGGCAGACTCTATCGTTGGCGGTCGCCCGTCATTGTGGAGAAAGGGGGTAAGGTTGAGGTCGAAGGTCACCTGATCGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCT；SEQ ID No.11；

FJ-Fu：AACCACAGCTCTCATATTCAGTTGATTTATAACTTAACGCTATGTGAGCTGAATGGCACAGATTGGCTGGCACAAAAATTTGACTGGGCAGTGGAGATTTTTGTCATCTTCCCCGTGTTGACTCACATTGTTTCCTATGGGGCACTCACCACCAGCCATTTCCTTGACACAGTTGGTCTGGCCACTGTGTCCACCGCCGGATATTATCACGGGCGGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCTCTGGCAGCGCTGATTTGCTTTGTCATTAGGCTTGCGAAGAACTGCATGTCCTGGCGCTACTCTTGTACCAGATATACCAACTTCCTTCTGGACACTAAGGGCAGACTTTATCGTTGGCGGTCACCCGTCATTGTGGAGAAAGGGGGTAAGGTTGAGGTCGAAGGTCACCTGATCGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCAACCCCT；SEQ ID No.12。

(2) DNA shuffling technique:

(21) PCR amplification was performed using the selected ORF5 gene of each group as a template, PCR products were analyzed by 1% gel electrophoresis, a band of interest was cut, and the DNA was recovered using a DNA purification recovery kit.

(22) And carrying out enzyme digestion on the recovered PCR product.

(23) Analyzing the enzyme digestion product by gel electrophoresis, observing a target band after enzyme digestion under an ultraviolet lamp of a gel imaging system, cutting the target band, and recovering by using a DNA purification recovery kit.

(24) Primer-free amplification of fragmented genes: and carrying out PCR amplification by taking the mixed segmented DNA as a template and a primer, analyzing a PCR product by gel electrophoresis, cutting a target band under an ultraviolet lamp of a gel imaging system, and recovering by using a DNA purification recovery kit.

(25) Amplification with primers: using the recovered product of the primer-free amplified fragment as a template, carrying out PCR amplification by using a specific primer, configuring 1% agarose gel for analyzing the PCR product, and recovering by using a DNA purification recovery kit. The specific primer sequences are as follows:

P1：CCGGAATTCATGAACAGCAGCTCAAATTTACAG；SEQ ID No.13；P2：CCGCTCGAGTTAAGGGGTAGCCGCGGAACCAT；SEQ ID No.14。

(3) and (3) connecting the PCR product recovered and purified in the step (25) to a PMD-19T vector for transformation, selecting 200 bacterial liquids containing recombinant plasmids, sending the bacterial liquids to Beijing Rui Boxing biological limited company for sequencing, analyzing the sequencing result by using DNAstar and MEGA 6.0 software for nucleotide and amino acid, and finally screening 2 fusion genes which simultaneously contain partial ORF5 genes of 4 pedigrees and are named as delta ORF5-8 and delta ORF 5-46.

The nucleotide sequence of Δ ORF5-8 is as follows:

AACAGCAGCTCAAATTTACAGTTGATTTATAACCTGACGATATGTGAGCTGAATGGCACAGACTGGCTGGACAAAAAATTTGATTGGGCAGTGGAGACTTTCGTCATCTTCCCTGTATTGACTCACATAGTCTCTTACGGCGCCCTCACCACTAGTCATTTTCTTGACACGGTCGGCCTGGCCACTGTGTCTACCGCCGGTTATTATCATGGGCGGTATGTGTTGAGTAGTATTTACGCTGTCTGTGCCCTGGCTGCGTTGGTTTGCTTCGCCATTAGGTTGGCGAAAAATTGCATGTCCTGGCGTTACTCATGCACCAGATATACTAATTTTCTCCTGGATACTAAGGGCACACTCTACCGCTGGCGGTCACCCGTCATCATTGAGAAAGGGGGTAAAGTTGATGTCGGGGGTCATTTAATCGACCTCAAAAGAGTTGTGCTTGATGGTTCCGCGGCTACCCCT；SEQ ID No.1。

the amino acid sequence encoded by Δ ORF5-8 is as follows:

NSSSNLQLIYNLTICELNGTDWLDKKFDWAVETFVIFPVLTHIVSYGALTTSHFLDTVGLATVSTAGYYHGRYVLSSIYAVCALAALVCFAIRLAKNCMSWRYSCTRYTNFLLDTKGTLYRWRSPVIIEKGGKVDVGGHLIDLKRVVLDGSAATP；SEQ ID No.3。

the nucleotide sequence of Δ ORF5-46 is as follows:

AACAGCAGCTCAAATTTACAGTTGATCTATAACCTGACGATATGTGAGCTGAATGGCACAGATTGGCTGGCACAAAAATTTGACTGGGCGGTGGAGATTTTTGTCATCTTCCCCGTGTTGACCCACATTGTTTCCTATGGGGCACTCACCACCAGCCATTTCCTTGACACAGTTGGTCTGGCCACTGTGTCCACTGCCGGATATTATCACGGGCGGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCTCTGGCAGCGCTGATTTGCTTTGTCATTAGGCTTGCGAAGAACTGCATGTCCTGGCGCTACTCTTGTACCAGATATACCAACTTTCTTCTGGACACTAAGGGCAAAATCTATCGTTGGCGGTCACCCGTCATCATAGAGAAAGAGGGTAAAGTTGAGGTCGGTGGCCATCTCATTGACCTCAGGAGAGTCGTGCTTGATGGTTCCGCGGCTACCCCT；SEQ ID No.2。

the amino acid sequence encoded by Δ ORF5-46 is as follows:

EXAMPLE 2 construction and transfection of recombinant plasmids

The fusion genes (delta ORF5-8 and delta ORF5-46) selected in example 1 and the eukaryotic expression vector PCAGGS-HA were digested with BamHI and XhoI, respectively, and ligated and transformed. Selecting positive bacteria liquid, sending to Beijing Rui Boxing biological limited company for sequencing, and adopting DNA star software to compare and analyze sequencing results. PCA-. DELTA.ORF 5-8 and PCA-. DELTA.ORF 5-46 were obtained.

MARC-145 cells were plated evenly on 6-well fine-pored cell culture plates at 37 ℃ with 5% CO one day prior to transfection experiments ₂ Culturing, and when the cells grow to about 80%, performing transfection according to the operation instruction of a kit (Lp8000 transfection kit).

Example 3 Indirect immunofluorescence and Western Blot detection

MARC-145 cells (1 cell slide per well) in 6-well transfected culture plates of example 2 were cultured for 48h and fixed with 4% paraformaldehyde, primary antibodies (PRRSV protein monoclonal antibody (MAb) (1:800 fold dilution), incubated at 37 ℃ for 1h, FITC-labeled goat anti-mouse IgG (1:1600 fold dilution) as secondary antibodies, washed with PBS, mounted on neutral resin, air dried, observed under a confocal laser microscope and photographed. The results are shown in FIG. 1. The results show that the transfected recombinant plasmid can detect specific green fluorescence in MARC-145 cells, and normal control cells have no specific fluorescence.

At the same time, MARC-145 cells transfected for 48h were sampled, RIPA protein lysate was added to dissolve the cell membrane, SDS-PAGE was performed and membrane-converted, and Western Blot detection was performed using mouse anti-HA protein antibody (diluted 1:800 fold) as the primary antibody and FITC-labeled goat anti-mouse IgG as the secondary antibody (diluted 1:1600 fold). The results are shown in FIG. 2. A sample of MARC-145 cells transfected with recombinant plasmids showed a band of interest at 17kDa, indicating that the two recombinant plasmids PCA- Δ ORF5-8 and PCA- Δ ORF5-46 were efficiently expressed in MARC-145.

Example 4 mouse immunization experiment

Plasmids used as vaccine for immunizing mice are extracted by an endotoxin-free large quality-improving particle kit. 56 female BALB/c mice 6-8 weeks old were randomly divided into 7 groups of 8 mice each, the first group was PBS blank control group, the second group was PCAGGS-HA empty vector group, the third group was attenuated vaccine group, the fourth group was PCA-ORF5-MLV (ORF5 gene from MLV vaccine) group, the fifth group was PCA- Δ ORF5-8 group, the sixth group was PCA- Δ ORF5-46 group, and the seventh group was PCA- Δ 2ORF5 (containing pedigree 1, 5 and 8 partial strain sequences). The PCA-ORF5-MLV group, PCA-Delta ORF5-8 group, PCA-Delta ORF5-46 group, PCA-Delta 2ORF5 group and PCAGGS-HA empty vector group each group immunized with 100. mu.g of endotoxin-free recombinant plasmid per medial thigh muscle of mice, PBS blank control group immunized with 100. mu.L of PBS per medial thigh muscle of mice, and attenuated vaccine group immunized with 100. mu.L of attenuated vaccine per medial thigh muscle of mice, all immunized 3 times with 14 days interval.

Wherein the nucleotide sequence of Δ 2ORF5 is as follows:

AACAGCAGCTCAcATTTACAGTTGATTTATAACTTGACGCTATGTGAGCTGAATGGCACAGATTGGCTGGCACAAAATTTTGACTGGGCAGTGGAGACTTTTGTCATCTTCCCCGTTTTGACTCACAtTGTTTCCTATGGGGCACTCACCACCAGCCATTTCCTTGACACAGTTGGTCTGatcACTGTGTCCACCGCCGGATATTATCACaGGCGGTATGTCTTGAGTAGCATTTACGCAGTCTGTGCCCTGGCTGCGTTGaCTTGCTTCGCCATTAGGTTGGTGAAAAATTGTATgTCCTGGCGCTACTCATGCACTAGATACACTAATTTTCTTCTGGATACTAAGGGCAAACTCTACCGTTGGCGGTCACCCGTCATCATAGAGAAGGGGGGTAAGGTTGAgGTTGAGGGTCATTTAATTGACCTCAAGAGAGTTGTGCTTGATGGTTCCGCGGCTACCCCT；SEQ ID No.15。

after two weeks (42d) and two weeks (56d) after the second immunization, whole blood of the mice is collected, and after serum is separated out, the GP5 protein antibody in the serum of the mice is detected by using an ELISA kit of PRRSV GP5 protein (PRRSV-GP 5). The results are shown in FIG. 3.

The results show that the PCA-delta ORF5-8, PCA-delta ORF5-46, PCA-ORF5-MLV, PCA-delta 2ORF5 and the attenuated vaccine can significantly enhance the level of specific antibodies induced in immunized mice (P <0.01) compared with the PCAGGS-HA empty vector group and the PBS blank control group. The PCA- Δ ORF5-46 group produced higher levels of GP5 antibody at 56d than the attenuated vaccine MLV group but less than the PCA-ORF5-MLV group compared to 42 d. The two recombinant DNA vaccines PCA-delta ORF5-8 and PCA-delta ORF5-46 are shown to stimulate the body to produce specific antibodies.

Example 5 serum neutralization assay

Mice were immunized as in example 4, and the experiment was carried out 14 days after the third immunization.

After inactivation of the collected murine sera, a fold-wise dilution was performed in combination with equal volumes of the FJ-Yang (lineage 1), FJ-Liu (lineage 3), FJ-Wang (lineage 5) and FJ-Fu (lineage 8.7) strains of 100TCID50, respectively, for seroneutralization experiments.

The neutralizing capacity of the immune mouse serum against PRRSV of different lineages was further evaluated by a method of fixing virus and diluting blood, and the results are shown in Table 1. 14 days after the third immunization, the sera of mice in the PCA-delta ORF5-46 group all produced higher levels of specific neutralizing antibodies against 4 different lineage strains, the average titer of neutralizing antibodies produced by FJ-Chen for lineage 1 was 12, the average titer of neutralizing antibodies produced by FJ-Liu for lineage 3 and FJ-Wang for lineage 5 was 11, and the average titer of neutralizing antibodies produced by FJ-Fu for lineage 8 was 16; the sera of mice in the PCA-delta ORF5-8 group were able to produce higher levels of specific neutralizing antibody titers against lineage 1, 3, and 8 representative strains, the mean titer of neutralizing antibodies produced by strain FJ-Chen was 16 for lineage 1, the mean titer of neutralizing antibodies produced by strain FJ-Liu was 10 for lineage 3, and the mean titer of neutralizing antibodies produced by strain FJ-Fu was 12 for lineage 8; while the serum of mice in the PCA-ORF5-MLV group represented high specific neutralizing antibodies produced by the strains to pedigrees 5 and 8 with average titers of 17 and 11, respectively, the serum of mice in the PCA-Delta 2ORF5 group represented high specific neutralizing antibodies produced by the strains to pedigrees 5 and 8 with average titers of 10 and 11, respectively, and the control group of immune vehicle and PBS failed to produce neutralizing antibodies, these results indicate that the PCA-Delta ORF5-8 and PCA-Delta ORF5-46 can induce the body to produce more effective neutralizing antibodies, especially for the newly circulating pedigree 1 and pedigree 3 strains.

TABLE 1 mouse serum neutralization assay

Note: "-" indicates no result.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Longyan college

<120> PRRSV ORF5 fusion gene DNA vaccine and preparation method

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

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<213> Artificial Sequence (Artificial Sequence)

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aacagcagct caaatttaca gttgatttat aacctgacga tatgtgagct gaatggcaca 60

gactggctgg acaaaaaatt tgattgggca gtggagactt tcgtcatctt ccctgtattg 120

actcacatag tctcttacgg cgccctcacc actagtcatt ttcttgacac ggtcggcctg 180

gccactgtgt ctaccgccgg ttattatcat gggcggtatg tgttgagtag tatttacgct 240

gtctgtgccc tggctgcgtt ggtttgcttc gccattaggt tggcgaaaaa ttgcatgtcc 300

tggcgttact catgcaccag atatactaat tttctcctgg atactaaggg cacactctac 360

cgctggcggt cacccgtcat cattgagaaa gggggtaaag ttgatgtcgg gggtcattta 420

atcgacctca aaagagttgt gcttgatggt tccgcggcta cccct 465

<210> 2

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

aacagcagct caaatttaca gttgatctat aacctgacga tatgtgagct gaatggcaca 60

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acccacattg tttcctatgg ggcactcacc accagccatt tccttgacac agttggtctg 180

gccactgtgt ccactgccgg atattatcac gggcggtatg tcttgagtag catttacgca 240

gtctgtgctc tggcagcgct gatttgcttt gtcattaggc ttgcgaagaa ctgcatgtcc 300

tggcgctact cttgtaccag atataccaac tttcttctgg acactaaggg caaaatctat 360

cgttggcggt cacccgtcat catagagaaa gagggtaaag ttgaggtcgg tggccatctc 420

attgacctca ggagagtcgt gcttgatggt tccgcggcta cccct 465

<210> 3

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

Asn Ser Ser Ser Asn Leu Gln Leu Ile Tyr Asn Leu Thr Ile Cys Glu

1 5 10 15

Leu Asn Gly Thr Asp Trp Leu Asp Lys Lys Phe Asp Trp Ala Val Glu

20 25 30

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

35 40 45

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

50 55 60

Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Ile Tyr Ala

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Val Cys Ala Leu Ala Ala Leu Val Cys Phe Ala Ile Arg Leu Ala Lys

85 90 95

Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe Leu

100 105 110

Leu Asp Thr Lys Gly Thr Leu Tyr Arg Trp Arg Ser Pro Val Ile Ile

115 120 125

Glu Lys Gly Gly Lys Val Asp Val Gly Gly His Leu Ile Asp Leu Lys

130 135 140

Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro

145 150 155

<210> 4

<211> 155

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Asn Ser Ser Ser Asn Leu Gln Leu Ile Tyr Asn Leu Thr Ile Cys Glu

1 5 10 15

Leu Asn Gly Thr Asp Trp Leu Ala Gln Lys Phe Asp Trp Ala Val Glu

20 25 30

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

35 40 45

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

50 55 60

Thr Ala Gly Tyr Tyr His Gly Arg Tyr Val Leu Ser Ser Ile Tyr Ala

65 70 75 80

Val Cys Ala Leu Ala Ala Leu Ile Cys Phe Val Ile Arg Leu Ala Lys

85 90 95

Asn Cys Met Ser Trp Arg Tyr Ser Cys Thr Arg Tyr Thr Asn Phe Leu

100 105 110

Leu Asp Thr Lys Gly Lys Ile Tyr Arg Trp Arg Ser Pro Val Ile Ile

115 120 125

Glu Lys Glu Gly Lys Val Glu Val Gly Gly His Leu Ile Asp Leu Arg

130 135 140

Arg Val Val Leu Asp Gly Ser Ala Ala Thr Pro

145 150 155

<210> 5

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gacagcagct cccatttaca gttgatctat aacctgacga tatgtgagct gaatggcaca 60

gattggcttg acaagaaatt tgactgggca gtggagactt tcgtcatctt ccctgtgctt 120

acccatattg tctcttacgg tgccctcacc accagccatt ttcttgacac ggtcggcctg 180

atcactgtgt ccaccgccgg ttattatcat gggcggtatg ttttgagtag catctacgct 240

gtctgtgccc tggctgcgtt tgtttgcttc accattaggt tggctaaaaa ttgcatgtcc 300

tggcgctact catgcaccag atatactaat tttcttctgg atactaaggg taaactctat 360

cgctggcggt catccgtcat catagagaaa gggggtaaag ttgatgttgg gggtcattta 420

atcgacctca agagagttgt gcttgatggt tccgcggcaa cccct 465

<210> 6

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gacagcagct cccacttaca attgatttat aacctaacga tatgtgagct gaatggcaca 60

gattggctga acaaaaaatt tgactgggca gtggagactt tcgttatttt ccctgtgttg 120

actcatattg tctcttacgg cgccctcacc actagccatt ttcttgacac ggttggcctg 180

atcactgtat ccaccgccgg ttactatcgt gggcggtatg tattgagtag catctacgct 240

gtctgtgccc tggctgcgtt tgtttgcttc gccattaggt tggcaaaaaa ttgtgtgtcc 300

tggcgctact catgcaccag atataccaat tttcttctgg atactaaggg caagctatat 360

cgctggcagt catccgtcat catagagaag gggggcaaag ttgatgtggg gggtcattta 420

atcgacctca agagagtcgt gcttgatggt tctgcggcaa cccct 465

<210> 7

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aacagcagct cccacttaca gtcgatttat aacctgacga tatgtgagct gaatggcaca 60

gattggctga acaaaaattt tgactgggca gtagagactt tcgttatttt tcctgtgctg 120

actcatattg tctcttacgg cgccctcacc actagccatt ttcttgacac aatcggcttg 180

atcactgtgt ccaccgccgg ttattatcac gggcggtatg tattgagtag catctacgct 240

gtctgtgccc tggctgcgtt cgtttgcttc gccattaggt tggcaaaaaa ttgtatgtcc 300

tggcgctact catgcaccag atataccaat tttcttctgg ataccaaggg caaactctat 360

cgctggcggt cacccgtcat catagagaaa gggggcaaag ttgatgttgg gggtcattta 420

atcgacctca agagagttgt gcttgatggt tccgcggcaa cccct 465

<210> 8

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aacagcagct cttattcgca gttgatttat aacctaacgc tatgtgagct gaatggcaca 60

gagtggctgg ccactagatt tgattgggca gtggaatgtt ttgtcatctt tcccgtgttg 120

actcacatca tctcctacgg tgccctcact accagccatt ttcttgacac aattagcctg 180

gtcactgtat ccaccgccgg gtattatcac gggcggtatg ttttgagtag tatctacgca 240

gtctgtgctt ttgctgcgtt aatctgcttt gtcattaggt tggcgaagaa ttgcatgtct 300

tggcgctgct catgtaccag gtatactaat tttcttcttg atactaaggg caaaatctat 360

cgttggcggt cacccgtcat catagagaaa gagggtaaag ttgaggtcgg tggccatctc 420

attgacctca agagagtcgt gcttgatggt tccgcggcaa ccctt 465

<210> 9

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aacagcagct cttattcgca gttgatttat aacctaacgc tatgtgagct gaatggcaca 60

gagtggctgg ccactagatt tgattgggca gtggaatgtt ttgtcatctt tcccgtgttg 120

actcacatca tctcctacgg tgccctcact accagccatt ttcttgacac agttagcctg 180

gtcactgtat ccaccgccgg gtattatcac gggcggtatg ttttgagtag tatctacgca 240

gtctgtgctt ttgctgcgtt aatctgcttt gtcattaggt tggcgaagaa ttgcatgtct 300

tggcgctact catgtaccag gtatactaat tttcttcttg atactaaggg caaaatctat 360

cgttggcggt cacccgtcat catagagaaa gagggtaaag ttgaggtcgg tggccatctc 420

attgacctca agagagtcgt gcttgatggt tccgcggcaa ccctt 465

<210> 10

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gacagcagct cccatctaca gctgatttac aacttgacgc tatgtgagct gaatggcaca 60

gattggctag ctaacaaatt tgattgggca gtggagagtt ttgtcatctt tcccgttttg 120

actcacattg tctcctatgg tgccctcact accagccatt tccttgacac agtcgcttta 180

gtcactgtgt ctaccgccgg gtttgttcac gggcggtatg tcctaagtag catctacgcg 240

gtctgtgccc tggctgcgtt gacttgcttc gtcattaggt ttgcaaagaa ttgcatgtcc 300

tggcgctacg cgtgtaccag atataccaac tttcttctgg acactaaggg cggactctat 360

cgttggcggt cgcctgtcat catagagaaa aggggcaaag ttgaggtcga aggtcatctg 420

atcgacctca aaagagttgt gcttgatggt tccgtggcaa cccct 465

<210> 11

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gacaacagct ctcatattca gttgatttat aacttaacgt tatgtgagct gaatggcaca 60

gattggctgg cacaaaaatt tgactgggca gtggagactt ttgtcatctt ccccgtgttg 120

actcacattg tttcctatgg ggcactcacc accagccatt tccttgacac agttggtctg 180

gccactgtgt ccaccgccgg atattatcac gggcggtatg tcttgagtag catttacgca 240

gtctgtgctc tggctgcgct gatttgcttt gtcattaggc ttgcgaagaa ctgcatgtcc 300

tggcgctact cttgtaccag atataccaac ttccttctgg acactaaggg cagactctat 360

cgttggcggt cgcccgtcat tgtggagaaa gggggtaagg ttgaggtcga aggtcacctg 420

atcgacctca agagagttgt gcttgatggt tccgcggcaa cccct 465

<210> 12

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aaccacagct ctcatattca gttgatttat aacttaacgc tatgtgagct gaatggcaca 60

gattggctgg cacaaaaatt tgactgggca gtggagattt ttgtcatctt ccccgtgttg 120

actcacattg tttcctatgg ggcactcacc accagccatt tccttgacac agttggtctg 180

gccactgtgt ccaccgccgg atattatcac gggcggtatg tcttgagtag catttacgca 240

gtctgtgctc tggcagcgct gatttgcttt gtcattaggc ttgcgaagaa ctgcatgtcc 300

tggcgctact cttgtaccag atataccaac ttccttctgg acactaaggg cagactttat 360

cgttggcggt cacccgtcat tgtggagaaa gggggtaagg ttgaggtcga aggtcacctg 420

atcgacctca agagagttgt gcttgatggt tccgcggcaa cccct 465

<210> 13

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ccggaattca tgaacagcag ctcaaattta cag 33

<210> 14

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ccgctcgagt taaggggtag ccgcggaacc at 32

<210> 15

<211> 465

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aacagcagct cacatttaca gttgatttat aacttgacgc tatgtgagct gaatggcaca 60

gattggctgg cacaaaattt tgactgggca gtggagactt ttgtcatctt ccccgttttg 120

actcacattg tttcctatgg ggcactcacc accagccatt tccttgacac agttggtctg 180

atcactgtgt ccaccgccgg atattatcac aggcggtatg tcttgagtag catttacgca 240

gtctgtgccc tggctgcgtt gacttgcttc gccattaggt tggtgaaaaa ttgtatgtcc 300

tggcgctact catgcactag atacactaat tttcttctgg atactaaggg caaactctac 360

cgttggcggt cacccgtcat catagagaag gggggtaagg ttgaggttga gggtcattta 420

attgacctca agagagttgt gcttgatggt tccgcggcta cccct 465

Claims

1. A PRRSV ORF5 fusion gene, which is characterized in that the fusion gene simultaneously contains partial ORF5 genes of 4 lineages;

the 4 lineages are lineage 1, lineage 3, lineage 5 subline 5.1, and lineage 8 subline 8.7;

the nucleotide sequence is as follows:

2. The PRRSV ORF5 fusion gene of claim 1, wherein Δ ORF5-8 encodes the amino acid sequence:

the amino acid sequence encoded by Δ ORF5-46 is as follows:

3. a porcine reproductive and respiratory syndrome vaccine comprising the fusion gene of any one of claims 1 to 2.

4. The porcine reproductive and respiratory syndrome vaccine of claim 3, constructed using the eukaryotic expression vector PCAGGS-HA.