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Recombinant antiviral protein as well as preparation method and application thereof

An anti-virus and protein technology, applied in the direction of anti-viral agents, peptide/protein components, recombinant DNA technology, etc., can solve the problems of unstable preventive effect, low specificity and high toxicity of chemical anti-viral drugs, and achieve high resistance Effects of PRRSV, high antiviral ability, and low toxicity

Inactive Publication Date: 2016-09-28
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a recombinant antiviral protein in order to overcome the defects in the prior art that the preventive effect of the vaccine against PRRSV is unstable, and the specificity of chemical antiviral drugs is not high and the toxicity is relatively high. And its preparation method and application

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  • Recombinant antiviral protein as well as preparation method and application thereof
  • Recombinant antiviral protein as well as preparation method and application thereof
  • Recombinant antiviral protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1 Construction of recombinant antiviral protein P9-ISG20 expression vector

[0053] In this example, a new clone P9-Linker-EGFP-Linker-ISG20 (P9-ISG20) was constructed, which fused P9 and ISG20 and was constructed on the pCDNA3.1(+) eukaryotic expression vector. For fluorescence detection experiments, the EGFP gene is connected to the N-terminal end of ISG20 through two linking sequences 3╳(GGGGS). The specific cloning design is as follows figure 1 shown. The build method is as follows:

[0054] 1) Construction of pcDNA-P9 plasmid vector

[0055] The P9 forward and reverse primers were annealed to construct the P9 gene, and the P9 forward primer and reverse primer were as follows (the nucleotide sequence of which is shown in SEQ ID No. 7-8 of the sequence table):

[0056] P9 forward primer:

[0057] 5'-AGCTTCACATCAGGCTGACATTGAGCAGAAATAAGAACACCG-3'

[0058] P9 reverse primer:

[0059] 5'-AATTCGGTGTTCTTATTTCTGCTCAATGTCAGCCTGATGTGA-3'

[0060] Use the ann...

Embodiment 2

[0078] Embodiment 2 Construction of recombinant antiviral protein TAT-P9-ISG20 expression vector

[0079] Using the pcDNA-P9-EGFP-ISG20 plasmid constructed in Example 1 as a template, design primers containing the TAT gene sequence, use a homologous recombination kit to connect TAT into the N-terminal of the P9-EGFP-ISG20 gene by homologous recombination, and amplify The amplification primers are as follows (the nucleotide sequence is shown in SEQ ID No.20-21 in the sequence table):

[0080] Upstream primers:

[0081] 5'-AAGCTTTATGGCAGGAAGAAGCGGAGACAGCGACGAAGACACATCA-3'

[0082] Downstream primers:

[0083]5'-GAATTCTCAGTCTGACACAGCCAGGCGGGGCAGCCCTCGGCGG-3'

[0084] The amplified TAT-P9-EGFP-ISG20 gene was purified and recovered, and the target gene TAT-P9-EGFP-ISG20 gene was cloned into the cell-free expression vector pIVEX-HisTag by the homologous recombination method in Example 1 to obtain the expression vector pIVEX TAT- P9-EGFP-ISG20. pIVEX-His Tag is an expression pla...

Embodiment 3

[0086] Example 3 Inhibition of recombinant antiviral protein P9-ISG20 on PRRSV virus infection

[0087] (1) Cultivate MARC-145 cells, spread the cells to 24-well cell culture plates, and wait until the cells grow to 70%;

[0088] (2) Transfect the recombinant antiviral protein P9-ISG20 expression vector constructed in Example 1, the P9 and ISG20 expression vectors and empty load as a control; the specific steps are as follows: 50 microliters of 1640 RIPM serum-free medium and 2 microliters of transfection Dyeing reagent (Liposome 2000) was mixed evenly. Mix 50 microliters of 1640 RIPM serum-free medium with 0.5 micrograms of expression plasmid vectors evenly. Mix the transfection reagent and the expression plasmid vector evenly, let stand at room temperature for 5 minutes, then directly add to the cell well, and continue to incubate in a 37°C incubator for 24 hours;

[0089] (3) 24 hours after transfection, the liquid in the cell wells was discarded, and the cells were washe...

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Abstract

The invention discloses a recombinant antiviral protein, nucleic acid coding the recombinant antiviral protein, a recombinant expression vector comprising the nucleic acid, a transformant comprising the recombinant expression vector, a method for preparing the recombinant antiviral protein and an application of the recombinant antiviral protein to preparation of medicines resistant to porcine reproductive and respiratory syndrome viruses (PRRSV). The recombinant antiviral protein comprises a P9 peptide at a terminal N and an ISG20 protein at a terminal C, wherein the P9 peptide and the ISG20 protein are covalently fused; an amino acid sequence of the P9 peptide is shown in SEQ ID NO.1. The recombinant antiviral protein can effectively inhibit synthesis of ribonucleic acid (RNA) of the PRRSV and block replication of the PRRSV, has very high antiviral ability and high target specificity and also has very low toxicity toward cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant antiviral protein and its preparation method and application. Background technique [0002] Porcine Reproductive and Respiratory Syndrome (PRRS) is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), which is one of the common diseases of domestic pigs. [0003] Previously, 15 peptides that can inhibit PRRSV polymerase or helicase have been obtained (Liu et al., 2012). Among these peptides, the P9 polypeptide (amino acid sequence: HRILMRIRQMMT) has a strong virus-inhibiting function by combining with viral polymerase. P9 is a peptide segment composed of 12 amino acids, with a short half-life, and will be rapidly metabolized and degraded in clinical applications. On the other hand, although P9 can bind to the polymerase of PRRSV, the mutation of the virus can greatly reduce the antiviral ability of p9. [0004] At this stage, the prevent...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/10A61P31/14
CPCA61K38/00C07K7/08C07K2319/00C12N9/22
Inventor 马志永刘珂侯凤香邵东华魏建超李蓓蓓邱亚峰史子学缪德年马改妮郇贝丽王飞飞
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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