Porcine alpha-interferon gene with efficient expression and high antiviral activity and application of expression protein thereof

A high-efficiency expression, alpha interferon technology, used in antiviral agents, medical preparations containing active ingredients, genetic engineering, etc., to achieve the effect of reducing economic losses

Active Publication Date: 2012-09-05
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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Problems solved by technology

Our research group also cloned pIFN-α into the eukaryotic expression vector pVAX1?, and the activity of pIFN-α expressed after transfection into HEK-293A cells was low, only 1×106.0 U / 0.1mL

Method used

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  • Porcine alpha-interferon gene with efficient expression and high antiviral activity and application of expression protein thereof
  • Porcine alpha-interferon gene with efficient expression and high antiviral activity and application of expression protein thereof
  • Porcine alpha-interferon gene with efficient expression and high antiviral activity and application of expression protein thereof

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Embodiment Construction

[0040]The porcine alpha interferon gene of the present invention will be further described below in combination with specific embodiments.

[0041] . eukaryotic plasmid pVAX1 ? Transformation into pMVAX1 ?

[0042] 1.1 Artificial synthesis of inserted genes

[0043] Refer to the Rabbit β-Globin Intron II gene sequence published by GenBank (GenBank No: V00882), and introduce it upstream Sst I enzyme cutting site and a sequence of pCMV immediate early promoter, introduce T7 promoter sequence and Eco The RI restriction site, this gene sequence was spliced ​​together and handed over to TaKaRa Company for artificial synthesis. The specific coded nucleotide sequence containing the Rabbit β-Globin Intron II gene is shown in sequence 1 in the sequence table, and cloned into the T vector.

[0044] A segment of the pCMV immediate early promoter sequence is 5'-GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGC-3',

[0045] The T7 promot...

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Abstract

The invention relates to the field of gene expression. The nucleotide sequence of a porcine alpha-interferon gene with efficient expression and high antiviral activity is shown as a sequence 2. Genes of the sequence 2 are cloned to a eukaryon expression vector to obtain a recombinant plasmid. A synthetic method of the porcine alpha-interferon gene comprises the following steps of: inserting a Rabbit beta-Globin Intron II, a pCMV immediate early promoter and a T7 promoter into pVAX1(C) to obtain pMVAX1(C); and inserting a maturation protein gene of pIFN-alpha into the pMVAX1(C) to obtain pMVAX1(C)-pIFN-alpha. The expression protein of the porcine alpha-interferon gene is applied to the preparation of a medicine for treating a porcine reproductive and respiratory syndrome. The porcine alpha-interferon gene with efficient expression and high antiviral activity breaks through the limitation that the expression amount is low and the activity is poor while being used for expressing the pIFN-alpha by the pMVAX1(C) carrier and the activity unit can be up to 2*10<7.0> U / 0.1 mL; and through the porcine alpha-interferon gene provided by the invention, the proliferation of PRRSV (Porcine Reproductive and Respiratory Syndrome Viruses) with high pathogenicity can be obviously inhibited and the economic loss of a pig farm can be reduced.

Description

technical field [0001] The invention relates to the field of gene expression in biotechnology, in particular to a porcine alpha interferon gene with high expression and high antiviral activity, and also relates to the application of the expressed protein. Background technique [0002] Interferon (IFN) is a class of cytokines with broad-spectrum anti-virus, anti-tumor, and immunomodulatory effects. IFN can be divided into two types (type I and type II). IFN-α belongs to type I interferon, which is the first line of defense for the body's cells against viral infection. It has three major functions: antiviral effect, antitumor effect and immune function. Regulatory effect. Lefevre et al first cloned the porcine IFN-α (pIFN-α) gene in 1990 and expressed its mature protein gene in Escherichia coli, which has strong antiviral activity. Subsequently, scholars at home and abroad expressed pIFN-α with adenovirus, Escherichia coli, yeast, etc., and conducted a lot of research on i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/21C12N15/79C12N15/10A61K38/21A61P31/14
Inventor 杜以军齐静王金宝吴家强张秀美
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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