Artificially synthesized polyepitope gene of porcine reproductive and respiratory syndrome virus and its application

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

CN103421817BActive Publication Date: 2016-01-13HUAZHONG AGRI UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2016-01-13

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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.
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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. Pigs are characterized by respiratory disease and high mortality. 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. 随后加拿大、德国、荷兰等一些国家也先后暴发了该病(BilodeauRetal,PorcinereproductiveandrespiratorysyndromeinQuebec.VetRec,1991,129:102-103;DeaS,BilodeauR,AthanassiousRetal.Swinerepr...

Examples

Embodiment 1

[0043] Embodiment 1 Design and artificial synthesis of highly pathogenic PRRSV antigen multi-epitope gene

[0044]The present invention is based on the highly pathogenic porcine reproductive and respiratory syndrome virus WUH3 strain (its nucleotide sequence comes from GenBank: HM853673.2, see literature: LiB, XiaoS, WangY, XuS, JiangY, ChenH, etal. .2009; 27(13):1957-1963) analysis of known epitopes, select B cell epitopes and T cell epitopes related to immune protection, connect each epitope gene in series with linker, and construct PRRSV antigen multi-epitope gene (mMEP). The selected epitopes include: the T1 cell epitope of GP3 (its amino acid sequence: LEPGKSFW, see the amino acid sequence described in SEQ ID NO: 3 in the sequence listing), the T2 cell epitope (its amino acid sequence: CRIGHDRCSEN, see the sequence listing SEQ ID NO: 4) The amino acid sequence described above); the T1 cell epitope of GP4 (its amino acid sequence: CLFAILLAI, see the amino acid sequence de...

Embodiment 2

[0046] Embodiment 2 Construction of pcDNA3.1-mMEP recombinant plasmid

[0047] 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).

[0048] 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 BamHI+NotI (see the enzyme digestion diagram figure 2 ), purify and recover a digested product of about 560bp, and combine it with the pcDNA3.1(+) eukaryotic expression plasmid digested with BamHI+NotI (see figure 2 ) connection, transformed Escherichia coli DH5α competent cells, extracted a small amount of plasmid (using the kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd., and operated according to...

Embodiment 3

[0050] Example 3 Large-scale preparation of plasmid DNA vaccine pcDNA3.1-mMEP and empty vector plasmid pcDNA3.1(+)

[0051] Use the large amount of plasmid extraction kit produced by Omega (operate according to the instructions in the kit) to carry out a large amount of plasmid (vaccine) extraction, the specific operation steps are as follows:

[0052] (1) Escherichia coli DH5α of the recombinant plasmid pcDNA3.1-mMEP or the empty vector plasmid pcDNA3.1(+) (purchased from Yingwei Jieji (Shanghai) Trading Co., Ltd.) were inoculated in 100 mL of ampicillin-resistant LB In liquid culture medium (the concentration of ampicillin is 100U / mL), culture with shaking overnight (12-16h);

[0053] (2) Transfer the Escherichia coli bacterial liquid obtained in step (1) to two 50mL centrifuge tubes, centrifuge at 12000r / min for 2min, discard the supernatant, and collect the bacterium precipitate;

[0054] (3) After resuspending the pellet with 2.5mL Solution I (included in the above kit),...