Modified pig propagation and respiratory syndrome virus ORF5 gene and use thereof

A technique for respiratory syndrome and pig breeding, applied in application, genetic engineering, virus antigen components, etc., can solve problems such as insufficient exposure of neutralizing epitopes and difficulty in stimulating neutralizing antibodies

Inactive Publication Date: 2006-05-31
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recently, Ostrouski et al. (Ostrowski M, Galeota JA, Jar AM, et al. Identification of neutralizing and nonneutralizing epitopes in the porcine reproductive and respiratory syndrome virus GP5 ectodomain. JVirol, 2002, 76: 4241~4250) identified in the use of phage surface display technology When GP5 neutralizes the epitope, it is found that there is a non-neutralizing epitope immediately upstream of the neutralizing epitope. This epitope has a coverage effect similar to that of the covered epitope in HIV, and it is the coverage effect of this covered epitope , resulting in the inability to fully expose the GP5 neutralizing epitope, making it difficult to elicit strong neutralizing antibodies

Method used

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  • Modified pig propagation and respiratory syndrome virus ORF5 gene and use thereof
  • Modified pig propagation and respiratory syndrome virus ORF5 gene and use thereof
  • Modified pig propagation and respiratory syndrome virus ORF5 gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Preparation of a plasmid containing the modified gene of the present invention and a plasmid containing a control gene

[0069] 1. Construction of eukaryotic plasmid pCI-52 expressing unmodified ORF5 gene (control)

[0070] Using pMD-ORF5 as a template, P52S and P52R as primers to amplify the ORF5 gene, the purified amplified product was digested with XhoI and XbaI, and cloned directly into the corresponding site of the eukaryotic expression vector pCI-neo to obtain the recombinant plasmid pCI- 52. Its structure is shown in Figure 2, and the results of enzyme digestion and PCR identification are shown in Figure 3 (XhoI and XbaI enzyme digestion only have a band of about 6000bp, XhoI+XbaI enzyme digestion produces a band of about 600bp and a band of about 5400bp, PCR amplified a band of about 600bp).

[0071] 2. Construction of the eukaryotic plasmid pCI-52M expressing the modified ORF5 gene

[0072] Using pMD-ORF5 as a template, use primers p51 and p5m1 to...

Embodiment 2

[0073] Embodiment 2: the biological experiment of DNA vaccine of the present invention and contrast vaccine to immune efficacy of mice

[0074] 1. Immunization procedure of Balb / c mice

[0075] Divide Balb / c mice into 3 groups, 6 mice in each group, and inject 100 μl (containing 100 μg plasmid) into each mouse by intramuscular injection of hind legs, and immunize 2 times with an interval of 2 weeks. As a negative control for nucleic acid immunization. At 2, 4, and 6 weeks after the first immunization, blood was collected through tail vein negative pressure, and the serum was separated to detect ELISA antibody and neutralizing antibody.

[0076] 2. ELISA antibody level

[0077] The GP5 protein expressed and purified by Escherichia coli was used as the antigen to detect the ELISA antibody level in the serum. The results showed that the ELISA antibody induced by the modified DNA vaccine pCI-52M was significantly higher than that of the unmodified DNA vaccine pCI-52M. 52 immune...

Embodiment 3

[0084] Embodiment 3: DNA vaccine of the present invention and control vaccine are to the biological experiment of weaned piglet immune efficacy

[0085] 1. Pig immunization program

[0086] The weaned piglets were randomly divided into 5 groups, 4 pigs in each group, and each pig was injected intramuscularly with 500 μL (containing 100 μg plasmid), and immunized 3 times with an interval of 2 weeks. control. Blood was collected through the anterior vena cava at 6 weeks, 8 weeks, and 10 weeks after the first immunization, and the serum was separated to detect the levels of ELISA antibodies and neutralizing antibodies. At the same time, anticoagulant blood was collected, and lymphocytes were separated to detect the level of cellular immune response.

[0087] 2. ELISA antibody detection

[0088] The GP5 protein expressed and purified by Escherichia coli was used as the antigen to detect the ELISA antibody level in the serum. The results showed that the ELISA antibody induced by ...

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Abstract

The invention belongs to the animal gene engineering technology field. It is about a gene and its application, the gene is modificatory porcine reproductive and respiratory syndrome virus ORF5. Its attribute is to insert nucleotide sequence between the neutralization and cover list of GP5, the nucleotide sequence is artificial synthesis coding accessorial T-lymph cell list. The sequence of this nucleotide is just as the sequence list of SEQ ID NO:1 and attached figure 1. This modificatory gene is included in eukaryotic expression plasmid, and the Escherichia coliDH5 / pCI-52M, which includes the plasmid, is conserved in CCTCC, and the number is CCTCC NO: M204080. This invention also presents the use of this gene in the preparation of pig bread and respiratory syndrome DNA vaccineíú

Description

technical field [0001] The invention relates to the technical fields of animal virology, animal infectious disease and genetic engineering. Specifically, it relates to the modification of porcine reproductive and respiratory syndrome virus ORF5 gene, and also relates to the immunogenicity evaluation after the gene modification and its application in new vaccines. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS for short, hereinafter referred to as PRRS) is a new viral infectious disease discovered in recent years. Disorders and respiratory disease and high mortality in pigs of all ages are characterized. The disease was first reported in the southern United States in 1987, and soon spread to the Midwest and spread rapidly across the United States. Subsequently, some countries such as Canada, Germany, and the Netherlands also successively broke out the disease (Bilodeau R et al, Porcine reproductive and respiratory...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/33A61K39/12C12N1/21C12N15/70C12N15/79
Inventor 方六荣陈焕春江云波肖少波金梅林吴斌刘正飞
Owner HUAZHONG AGRI UNIV
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