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Synthetic mMEP of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) and application thereof

A technique for respiratory syndrome and artificial synthesis, applied in application, gene therapy, antiviral agent, etc.

Active Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

YANG et al. reported three discontinuous epitopes of the M protein (EpORF6A, EpORF6B, EpORF6C), but only monoclonal antibodies to EpORF6A and EpORF6B showed neutralizing virus activity

Method used

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  • Synthetic mMEP of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) and application thereof
  • Synthetic mMEP of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) and application thereof
  • Synthetic mMEP of PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 Design and artificial synthesis of highly pathogenic PRRSV antigen multi-epitope gene

[0043]The present invention detects highly pathogenic porcine reproductive and respiratory syndrome virus WUH3 strain (its nucleotide sequence comes from GenBank: HM853673.2, see literature: LiB, Xiao S, Wang Y, Xu S, Jiang Y, Chen H, et al.Immunogenicity of the highly pathogenic porcine reproductive and respiratory syndrome virus GP5protein encoded by a synthetic ORF5gene.Vaccine.2009;27(13):1957-1963) Analysis of known epitopes, selection of B cell epitopes related to immune protection and T cell epitopes, each epitope gene was connected in series with a linker to construct a PRRSV antigen multi-epitope gene (mMEP). The selected epitopes include: T1 cell epitope of GP3 (its amino acid sequence: LEPGKSFW, see the amino acid sequence described in the sequence listing SEQ ID NO: 3), T2 cell epitope (its amino acid sequence: CRIGHDRCSEN, see the sequence listing SEQ ID NO...

Embodiment 2

[0045] Embodiment 2 Construction of pcDNA3.1-mMEP recombinant plasmid

[0046] The construction, preparation, and enzyme digestion analysis of the plasmid were all carried out according to conventional methods (see: J. Sambrook, EF Fritsch, T Maniartis, translated by Huang Peitang, Wang Jiaxi, etc., Molecular Cloning Experiment Guide (Third Edition) ), Science Press, 2002 edition).

[0047] The specific construction steps are: after the original vector pGH-mMEP containing the highly pathogenic PRRSV antigen multi-epitope gene mMEP in Example 1 is digested with BamH I+Not I (see figure 2 ), purify and recover a digested product of about 560bp, and combine it with the pcDNA3.1(+) eukaryotic expression plasmid digested with BamH I+Not I (refer to figure 2 ) connection, transform Escherichia coli DH5α competent cells, extract a small amount of plasmid (using the kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd., operate according to the instructions of the kit)...

Embodiment 3

[0049] Example 3 Large-scale preparation of plasmid DNA vaccine pcDNA3.1-mMEP and empty vector plasmid pcDNA3.1(+)

[0050] Use the large amount of plasmid extraction kit produced by Omega (operate according to the instructions in the kit) for large amount of plasmid (vaccine) extraction. The specific operation steps are as follows:

[0051] (1) Escherichia coli DH5α of recombinant plasmid pcDNA3.1-mMEP or empty vector plasmid pcDNA3.1(+) (purchased from Yingwei Jieji (Shanghai) Trading Co., Ltd.) were inoculated in 100mL LB containing ampicillin resistance In liquid culture medium (the concentration of ampicillin is 100U / mL), culture with shaking overnight (12-16h);

[0052] (2) Transfer the E. coli liquid obtained in step (1) to two 50mL centrifuge tubes, centrifuge at 12000r / min for 2min, discard the supernatant, and collect the bacterial precipitate;

[0053] (3) After resuspending the pellet with 2.5mL Solution I (included in the above kit), add 2.5mL Solution II (includ...

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Abstract

The invention relates to the technical field of animal virology, epizootiology and genetic engineering, in particular to a synthesis method and application of an mMEP of a PRRSV (Porcine Reproductive and Respiratory Syndrome Virus) with high pathogenicity. The synthesis method is characterized in that the T-cell epitopes of the GP3, GP4, GP5, protein M and protein N of a PRRSVWUH3 strain are connected with the epitope of a modified GP5B cell through linkers; according to mammal codon preference, codons are modified to synthesize the mMEP artificially. The nucleotide sequence of the mMEP is shown in SEQ ID NO:1; the protein sequence of the mMEP is shown in SEQ ID NO:2. The synthetic mMEP is contained in an eukaryotic expression plasmid pcDNA3.1-mMEP. Escherichia coli DH5(Alpha) / pcDNA3.1-mMEP containing the eukaryotic expression plasmid pcDNA3.1-mMEP are preserved in China Center for Type Culture Collection (CCTCC), and the preservation number of the escherichia coli DH5(Alpha) / pcDNA3.1-mMEP is CCTCC NO: M 2012171. The invention further discloses application of the mMEP in preparing an PRRS DNA vaccine.

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 and synthesis of highly pathogenic porcine reproductive and respiratory syndrome virus antigen multi-epitope gene, and also relates to the application of the modified multi-epitope gene in preparing porcine reproductive and respiratory syndrome DNA vaccine. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS, hereinafter referred to as PRRS) is a new viral infectious disease discovered in recent years. and respiratory disease and high mortality in pigs of all ages. 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, Porcinereproductive and...

Claims

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Application Information

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IPC IPC(8): C12N15/40C12N1/21A61K48/00A61P31/14C12R1/19
Inventor 方六荣肖少波王荡罗锐吴群峰李振曾松林
Owner HUAZHONG AGRI UNIV