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DNA (deoxyribonucleic acid) level-based highly pathogenic blue-eared pig disease JEV (Japanese encephalitis virus) replicon vaccine and application thereof

A porcine blue-ear disease virus, highly pathogenic technology, applied in the field of genetic engineering, can solve the safety hazards of host genome recombination with a large vaccination dose, and achieve the effect of high antibody and strong lymphocyte proliferation response

Inactive Publication Date: 2011-11-23
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although DNA vaccines have many advantages, they are subject to many restrictions in the development due to the large inoculation dose and the potential safety hazard of recombination with the host genome.

Method used

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  • DNA (deoxyribonucleic acid) level-based highly pathogenic blue-eared pig disease JEV (Japanese encephalitis virus) replicon vaccine and application thereof
  • DNA (deoxyribonucleic acid) level-based highly pathogenic blue-eared pig disease JEV (Japanese encephalitis virus) replicon vaccine and application thereof
  • DNA (deoxyribonucleic acid) level-based highly pathogenic blue-eared pig disease JEV (Japanese encephalitis virus) replicon vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of Replicon Vaccine for Porcine Highly Pathogenic PRRS

[0027] 1. The design of constructing a replicon vaccine for porcine highly pathogenic PRRS (as shown in the structure diagram figure 1 )

[0028] Through self-designed primers (shown in SEQ ID NO: 19-29), using the reverse transcription product of HP PRRS gene RNA as a template, primers GP5F and GP5R-FMDV2A-R can amplify the complete coding region of the GP5 gene and The first 45 bases of the 2A sequence, the result is a specific fragment of 648bp in size; the primers MF-FMDVF2A-F and MR-SalI can amplify the last 45 bases of the 2A sequence and the complete coding region of the M gene , 570bp was amplified. In addition, the primers GP5F and MR-SalI amplified a G-2A-M fragment of approximately 1,200 bp by fusion PCR. At the same time, G-2A-M fragment was used as template, GP5F and MR were used as primers to amplify G-2A-MR fragment of about 1,200 bp, and then IRES1F and IRES-588R were used as ...

Embodiment 2

[0033] Example 2 Detecting the expression of GP5 protein and M protein by Western-blot with chemiluminescence method

[0034] The plasmids co-expressing GP5 and M proteins of PRRSV (pJEV-REP-G-2A-M and pJEV-REP-G-2A-M-IRES) and eukaryotic expression plasmids (pCAGGS-GM) were respectively transfected into 293T cells, Cells were collected 48 hours after transfection, and 48 hours after recombinant plasmid transfection, cells were harvested, washed twice with PBS, resuspended in appropriate amount of PBS, added 2×SDS loading buffer, and left in boiling water bath for 10 minutes. A 12% SDS-polyacrylamide gel (PAGE) was prepared, spotted at 20μl per well, and electrophoresed at 80V / 0.5h and 120V / 1.5h. After the electrophoresis, the protein was transferred to the nitrocellulose membrane under the condition of 17V / 10min on a semi-dry electrotransfer machine. After the transfer was completed, the membrane was washed with 1×PBS for 5 minutes, and blocked with 5% skim milk at 37°C for 1 ...

Embodiment 3

[0035] Example 3 Indirect immunofluorescence detection of JEV non-structural protein expression

[0036] The JEV replicon plasmids (pJEV-REP-G-2A-M and pJEV-REP-G-2A-M-IRES) co-expressing the GP5 and M proteins of PRRSV and the vector control plasmid (pJEV-REP-IRES) were respectively transformed After 48 hours of infecting 293T cells, it was detected by indirect immunofluorescence that all three JEV replicon recombinant plasmids could express JEV NS1 protein (see Figure 5 ).

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Abstract

The invention discloses a DNA (deoxyribonucleic acid) level-based highly pathogenic blue-eared pig disease JEV (Japanese encephalitis virus) replicon vaccine and an application thereof. In the invention, primer amplification is carried out to obtain GP5 and M genes of a highly pathogenic blue-eared pig disease virus XH strain, an FMDV-2A sequence is inserted between the two genes by utilizing a fusion PCR (polymer chain reaction) method to obtain a G-2A-M segment, and finally the G-2A-M segment is cloned into pJEV-REP by utilizing two restriction enzyme cutting sites SpeI and SalI; besides, an IRES (internal ribosome entry site) sequence (G-2A-M-IRES) is inserted into the downstream of the G-2A-M segment, thus M protein can produce a real N terminal; and the G-2A-M-IRES segment is inserted into the pJEV-REP by utilizing two restriction enzyme cutting sites SalI-HF and SpeI, and finally a highly pathogenic blue-eared pig disease vaccine which is based on a JEV replicon and can express GP5 and M proteins is constructed.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a highly pathogenic porcine PRRS virus JEV replicon vaccine based on DNA level and application thereof. Background technique [0002] PRRS vaccines currently in clinical use include inactivated vaccines and live attenuated vaccines. Due to the unsatisfactory immune effect of inactivated vaccines, and the possibility of strong virulence in live attenuated vaccines. [0003] At present, PRRS has become one of the major infectious diseases that seriously harm the pig industry, and vaccination is still the most effective measure to prevent and control PRRS. At present, the commercialized vaccines used to prevent PRRS are mainly attenuated vaccines and inactivated vaccines, but due to safety hazards or poor immune effects, they cannot provide ideal immune protection. The DNA vaccine, known as the "third vaccine revolution", puts the gene encoding a certain antigenic prot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K39/12C12N15/85A61P31/14
Inventor 廖明陈孝明亓文宝臧富玉李红梅
Owner SOUTH CHINA AGRI UNIV
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