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New porcine reproductive and respiratory syndrome virus ORF5 modified gene and application thereof

A respiratory syndrome and ORF5 technology, applied in the field of molecular biology, can solve the problems of unsatisfactory GP5 gene immune protection efficiency, interference, and inability to produce strong neutralizing antibodies.

Active Publication Date: 2012-01-18
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

E protein is a transmembrane glycoprotein located on the envelope of virions, which is an important protective antigen protein, but it cannot produce strong neutralizing antibodies, and the immune protection efficiency of GP5 gene is not ideal. The possible main reason is Yes: (1) There are neutralizing epitopes and non-neutralizing epitopes in the GP5 protein, and the non-neutralizing epitopes interfere with the neutralizing epitopes; (2) The glycosylation site on the GP5 protein can also make the virus Immune evasion occurs, thereby reducing the level of antibodies produced; (3) The gene expression efficiency in pig somatic cells is relatively low, and the antigen presentation effect is relatively weak. Therefore, in order to improve the immune efficacy of gene vaccines, it is necessary to carry out these three aspects. Comprehensive consideration, design of new antigen molecules

Method used

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  • New porcine reproductive and respiratory syndrome virus ORF5 modified gene and application thereof
  • New porcine reproductive and respiratory syndrome virus ORF5 modified gene and application thereof
  • New porcine reproductive and respiratory syndrome virus ORF5 modified gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction and Identification of Porcine Reproductive and Respiratory Syndrome Virus ORF5 Gene Modification and Its Eukaryotic Expression Plasmid

[0039] 1.1 Primer design

[0040] According to the GP5 gene sequence of PRRSV SY0608 strain (GenBank::EU144079.1), a pair of specific primers SY GP5.1 and SY GP5.2 were designed and synthesized, and pShuttle-GP5 plasmid (Li Yufeng, Jiang Ping, et al. Porcine Reproduction and Respiration Construction and immunogenicity determination of recombinant adenovirus with GP5 protein of syndrome virus [J]. Chinese Virology, 2006, 21(4): 364-367) as template to amplify GP5 gene.

[0041] Upstream primer SY GP5.1 5'-ATTCTCGAGATGTTGGGGAAGTGC-3' (SEQ ID NO.6),

[0042] The downstream primer SY GP5.2 5'-CGATCTAGACTAGAGACGACCCCATT-3' (SEQ ID NO.7), introduced restriction enzyme sites XhoI and XbaI at the 5' ends of the upstream and downstream primers respectively. The above primers were provided by Shanghai Yingjun Biotechnolog...

Embodiment 2

[0054] Example 2 Study on immune characteristics of eukaryotic plasmid DNA mice expressing modified GP5 protein

[0055] 2.1 Mass preparation of eukaryotic expression plasmids

[0056] A large number of plasmids were extracted by alkaline cleavage method, OD260 and OD280 were measured with a spectrophotometer, and the content and purity of each recombinant eukaryotic plasmid were calculated. DNA content (μg / mL) = sample dilution factor × 50 × OD260.

[0057] 2.2 Grouping of mice and immunization experiments

[0058] Seventy female Balb / c mice aged 6-8 weeks were divided into 7 groups with 10 mice in each group. The first group is the negative control group, injected with PBS, and the second, third, fourth, fifth, sixth, and seventh groups were injected with pCI-neo, pCI-GP5, pCI-SynGP5, pCI-SynGP5 / GP4-5, pCI-SynGP5 / GP4-5, The dose of pCI-SynGP5 / N-7 and pCI-SynGP5 / GP4-3 was 100 μg per mouse, and the same dose was boosted with the same dose on the 21st and 42nd days after the...

Embodiment 3

[0068] Example 3 Eukaryotic plasmid expressing modified GP5 protein and its recombinant attenuated Salmonella immune characteristics of piglets

[0069] 3.1 Construction and identification of recombinant attenuated Salmonella

[0070] Prepare competent cells of attenuated Salmonella SL3263 (attenuated strain of Salmonella choleraesuis C500, China Veterinary Drug Administration, the same below), and then transfer the recombinant plasmid into the attenuated Salmonella SL3263 by electroporation, after transformation, coat the transformed product LB plates containing 50 μg / mL ampicillin resistance were cultured overnight at 37°C. Pick a single transformed colony and culture it in LB liquid medium containing 50 μg / mL ampicillin until OD600nm=1.0; extract the plasmid by alkaline cleavage method, and then identify it by double enzyme digestion, which proves that the recombinant attenuated Salmonella SL-pCI-SynGP5 was obtained / GP4-5( Figure 9 ).

[0071] 3.2 Pig immune and challe...

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Abstract

The invention belongs to the field of molecular biology, and discloses a new porcine reproductive and respiratory syndrome virus ORF5 modified gene and an application thereof. The porcine reproductive and respiratory syndrome virus ORF5 modified gene has a sequence as shown in SEQ IDNO. 2, SEQ IDNO. 3, SEQ IDNO. 4, or SEQ IDNO. 5. A recombinant plasmid containing the porcine reproductive and respiratory syndrome virus ORF5 modified gene is provided. Attenuated salmonella SL-pCI-SynGP5 / GP4-5 containing the recombinant plasmid is provided. Results show that the constructed recombinant plasmid can induce high-level humoral immune response, lymphocyte proliferation response, and cytokine response. the recombinant salmonella can induce high-level antibody response, and induce high-level interferon; the viremia level is low; the damage of tissues and organs after toxin attack is relatively slight, and the biosafety level is high.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a novel porcine reproductive and respiratory syndrome virus ORF5 modified gene and application thereof. technical background [0002] Porcine reproductive and respiratory syndrome virus (PRRSV) GP5 protein (also known as E protein) is an envelope protein encoded by ORF5 and is a major structural protein of the virus. E protein is a transmembrane glycoprotein located on the envelope of virions. It is an important protective antigen protein, but it cannot produce strong neutralizing antibodies, and the immune protection efficiency of GP5 gene is not ideal. The possible main reason is Yes: (1) There are neutralizing epitopes and non-neutralizing epitopes in the GP5 protein, and the non-neutralizing epitopes interfere with the neutralizing epitopes; (2) The glycosylation site on the GP5 protein can also make the virus Immune evasion occurs, thereby reducing the level of antibodies prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/63C12N1/21A61K48/00A61K39/12A61P31/14C12R1/42
Inventor 姜平华莉李玉峰
Owner NANJING AGRICULTURAL UNIVERSITY
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