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Modified ORF2 gene of toroidal virus of pig, and application

A porcine circovirus, gene technology, applied in application, gene therapy, genetic engineering and other directions, can solve the problem of low level of neutralizing antibody and cellular immunity

Inactive Publication Date: 2007-12-26
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above studies all found that the level of neutralizing antibody and cellular immunity induced by DNA vaccine expressing ORF2 protein was not high

Method used

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  • Modified ORF2 gene of toroidal virus of pig, and application
  • Modified ORF2 gene of toroidal virus of pig, and application
  • Modified ORF2 gene of toroidal virus of pig, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Preparation of a plasmid containing the modified gene of the present invention and a plasmid containing a control gene

[0063] 1. Construction of eukaryotic plasmid pcORF2 expressing unmodified ORF2 gene

[0064] Using pT-PCV as a template, P3 and P4 as primers to amplify the ORF2 gene, recover and purify the amplified product, digest it with Hind III and Sal I, and clone it directly into the eukaryotic expression vector pcDNA3. Between the Hind III and Sal I sites of 1(+), the recombinant plasmid pcORF2 was obtained. Its structure is shown in Figure 1.

[0065] 2. Construction of the eukaryotic plasmid pcDNA-spORF2Δ41TM expressing the modified ORF2 gene

[0066] The ORF2 gene was amplified by PCR with primers P8 and P17, and the ORF2Δ41 gene fragment was obtained, which lacked the 41 amino acids at the N-terminal of the ORF2 gene and the terminator. +) Between the EcoR V and Not I sites (purchased from Invitrogen, USA), the recombinant plasmid pcORF2Δ41 ...

Embodiment 2

[0067] Embodiment 2: The biological experiment of pig weaning multiple systemic wasting syndrome DNA vaccine of the present invention and contrast vaccine to mouse immune efficacy

[0068] 1) Immunization procedure for Balb / c mice

[0069] The Balb / c mice were divided into 3 groups, respectively the porcine weaning multiple systemic wasting syndrome DNA vaccine pcDNA-spORF2Δ41TM of the present invention, the pcORF2 immunization group and the blank vector pcDNA3.1 (+) control group, 6 mice in each group, using Intramuscular injection into hind legs, 100 μl per mouse (containing 100 μg plasmid), immunized twice with an interval of 2 weeks. At 2, 4, and 6 weeks after the first immunization, blood was collected through tail vein negative pressure, serum was separated, and neutralizing antibodies were detected. Six weeks after the first immunization, all immunized mice were sacrificed through the cervical spine, the spleen was aseptically removed, and spleen lymphocytes were separ...

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Abstract

This invention relates to modified ORF2 gene of porcine circovirus type 2 and its application. The modified ORF2 gene is obtained by: replacing the gene coding 41 amino acids at N-termal of natural ORF2 with the gene coding signal peptide of human tissue plasminogen activator, deleting terminator of ORF2 gene, and fusing the gene coding C-terminal of ORF2 with the gene coding the transmembrane peptide of avian influenza virus HA protein. The nucleotide sequence of ORF2 gene is shown in SEQ ID No.1. This invention also relates to eukaryotic plasmid pcDNA-spORF2 Delta41TM, and host E. coli strain DH5alpha / pcDNA-spORF2 Delta 41TM (CCTCC No.M207069). The ORF2 gene and the plasmid can be used for manufacturing the vaccine for preventing post-weaning multi-systematic syndrome.

Description

technical field [0001] The invention relates to the technical fields of animal virology and genetic engineering. Specifically, it relates to the modification of a porcine circovirus type 2 ORF2 gene, and also relates to the immunogenicity evaluation after the gene modification and its application in a novel DNA vaccine. Background technique [0002] Porcine circovirus type 2 (PCV2 for short) is the main pathogen that causes porcine post-weaning multisystemic wasting syndrome (Post-weaning multisystemic syndrome, PMWS). The disease was first discovered in western Canada in 1991 (Clark EG. Post-weaning multisystemic wasting syndrome. Proc Am Assoc Swine Pract, 1997, 28: 499-501). Its clinical symptoms mainly include progressive emaciation, respiratory symptoms, and fever. and jaundice etc. Subsequently, the disease appeared successively in the United States, France, Spain, Northern Ireland and other countries or regions (Daft B et al. ; Kennedy S et al. Porcine circovirus i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/33C12N15/79A61K48/00
Inventor 肖少波樊惠英方六荣江云波谢立兰陈焕春
Owner HUAZHONG AGRI UNIV
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