Composite porcine alpha-IFN gene and recombinant vector thereof

A technology of α-interferon and gene, which is applied in the field of compound pig α-interferon gene sequence and its recombinant vector, can solve the problems of large loss of recombinant protein, high cost, limited clinical and scientific application, etc., and achieve the increase of expression Effect

Inactive Publication Date: 2010-09-29
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] The production of porcine IFN-α induced by inducers is expensive due to many limitations such as few sources, high cost, and complicated purification process, which greatly limits clinical and scientific applications.
In the prokaryotic expression system, its products mainly exist in the form of inclusion bodies without biological activity. If you wa...

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  • Composite porcine alpha-IFN gene and recombinant vector thereof
  • Composite porcine alpha-IFN gene and recombinant vector thereof
  • Composite porcine alpha-IFN gene and recombinant vector thereof

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Embodiment Construction

[0047] 1. Test materials

[0048] Escherichia coli DH5a, yeast X-33 and pPICZα-A vectors were purchased from Shanghai Yingjun Company; chemical reagents and kits used were purchased from Takara Company; Phenyl Sepharose TM 6Fast Flow and Q Sepharose FastFlow were purchased from Shanghai GE Company; PK-15 cells (pig kidney cells), VSV (vesicular stomatitis virus), PRV (porcine pseudorabies virus) and pig natural alpha interferon are all publicly known (Cao Ruibing et al., A new porcine α-interferon gene clone and its expression in Escherichia coli, Journal of A agricultural Biotechnology 2004, 12 (3): 278~282).

[0049] 2. Experimental design and production method

[0050] (1) Design and amplification of the gene sequence of a novel composite porcine interferon-alpha recombinantly inserted into Pichia pastoris

[0051] (A) Search the gene sequence of porcine interferon alpha on GenBank, and select 27 subtypes of wild type and strong disease resistance as templates;

[0052]...

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Abstract

The invention relates to a composite porcine alpha-IFN gene and a recombinant vector thereof, and belongs to the technical field of genetic engineering biological products. The newly designed gene sequences of composite porcine alpha-IFN are recombined to pPICZ alpha-A vectors, and the vectors are electronically transformed into yeast competent cells; and the positive identification, and the screening and the induced expression of high copy yeast strains are carried out. A pichia pastoris expression system is used for expressing foreign genes so as to be favorable for the commercial production of porcine IFN. Compared with the natural IFN which is expressed by yeast, the expression level of the protein which is expressed by genome increases by 3.17 times. The fluorescent quantitative PCR result shows that the level of PRVmRNA after PK-15 cells treated by 10-6mg/ml of the composite porcine alpha-IFN are re-infected with PRV is obviously lower than the level of PRVmRNA after the PK-15 cells which are not treated by the composite porcine alpha-IFN are infected with the PRV, and the level of PRVmRNA decreases by 37 times.

Description

1. Technical field [0001] The invention discloses a gene sequence of a compound porcine alpha interferon and its recombinant carrier, belonging to the technical field of genetic engineering biological products. Compared with natural porcine interferon alpha, it has stronger antiviral activity and can be used for the prevention and early treatment of various viral diseases in pigs. 2. Background technology [0002] Interferon is one of the most important cytokines in the animal body. It has broad-spectrum and high-efficiency anti-virus functions and plays a key role in regulating the immune system. As early as 1986, Lefevre et al. had done detailed research on porcine alpha interferon gene and porcine alpha interferon. A fragment containing α-interferon gene was obtained. Sequence analysis showed that the fragment had a continuous open reading frame containing 190 codons, a 5' region of about 100 nucleotides upstream of the initiation codon, and a TATTTAA sequence ( It has ...

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Application Information

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IPC IPC(8): C12N15/21C12N15/81C12R1/84
Inventor 黄丽陈溥言刘珂茅翔魏建超曹瑞兵王秀花侯凤香冯秀丽周斌王凤娟张丽颖
Owner NANJING AGRICULTURAL UNIVERSITY
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