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Efficiently expressed series porcine alpha and gamma interferon genes and application of expressed protein thereof

A high-efficiency expression technology of gamma interferon, applied in the biological field, to achieve the effect of reducing economic losses

Active Publication Date: 2012-10-24
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Our research group also cloned pIFN-γ into the eukaryotic expression vector pVAX1?, and the activity of pIFN-γ expressed after transfection into HEK-293A cells was low, only 1×105.0 U / 0.1mL

Method used

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  • Efficiently expressed series porcine alpha and gamma interferon genes and application of expressed protein thereof
  • Efficiently expressed series porcine alpha and gamma interferon genes and application of expressed protein thereof
  • Efficiently expressed series porcine alpha and gamma interferon genes and application of expressed protein thereof

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Experimental program
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Embodiment Construction

[0048] . eukaryotic plasmid pVAX1 ? Transformation into pMVAX1 ?

[0049] 1.1 Artificial synthesis of inserted genes

[0050] Rabbit β-Globin Intron II gene sequence published by GenBank (GenBank No: V00882), introduced upstream Sst I enzyme cutting site and a sequence of pCMV immediate early promoter, introduce T7 promoter sequence and Eco RI restriction site, a segment of the pCMV immediate early promoter sequence is 5'-GTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGC-3',

[0051] The T7 promoter sequence is 5'-AATACGACTCACTATAGGG-3';

[0052] This gene sequence was spliced ​​together and handed over to TaKaRa Company for artificial synthesis. The artificially synthesized nucleotide sequence is shown as sequence 1 in the sequence listing, and this gene sequence was cloned into the T vector.

[0053] 1.2 pMVAX1 ? construction and identification of

[0054] from the T carrier Sst I / Eco The target gene was cut with RI...

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Abstract

The invention relates to the field of gene expression, in particular to efficiently expressed series porcine alpha and gamma interferon genes. The nucleotide sequence of the interferon genes is shown as a sequence 2. The genes of the sequence 2 are cloned to eukaryotic expression vectors to obtain recombinant plasmids. A synthesis method of the series porcine alpha and gamma interferon genes comprises the following steps of: inserting Rabbit beta-GlobinIntronII, a pCMV immediate early promoter and a T7 promoter into pVAX1 to obtain pMVAX1, and inserting fusion protein genes pIFN-alpha / gamma into pMVAX1 to obtain pMVAX1-pIFN-alpha / gamma. The invention also relates to application of expression protein of the interferon genes in preparation of a medicament for treating porcine reproductive and respiratory syndrome. Through the interferon genes, the limitations of low expression quantity and poor activity when pIFN-alpha and pIFN-gamma are expressed through the pVAX1 are broken, the activity reaches 1*10<8.0>U / 0.1mL, the multiplication of high pathogenic porcine reproductive and respiratory syndrome viruses (PRRSV) can be obviously inhibited, and the economic loss of a pig farm is reduced.

Description

technical field [0001] The invention relates to gene expression in the field of biotechnology, in particular to a high-efficiency-expressed porcine alpha and gamma interferon in tandem, and also relates to the use of the coding gene of the interferon and its 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 Escherichia coli, adenovirus, yeast, etc., and conducted ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/79C12N15/10A61K38/21A61P31/14
Inventor 王金宝杜以军吴家强齐静张秀美李俊孙文博丛小燕时建立
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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