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Recombinant pig alpha-interferon as well as preparation method and application thereof

A technology of interferon and restriction endonuclease, applied in the field of preparation of recombinant porcine α-interferon, can solve the problems of no cytotoxicity found, achieve good market potential, low endotoxin content, and improve safety

Inactive Publication Date: 2018-06-19
QINGDAO VLAND BIOTECH INC +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Porcine α-interferon has a certain effect on infectious gastroenteritis, epidemic diarrhea, and blue ear disease in pigs, but it did not find that other drugs were used at the same higher concentration when using α-interferon in the virus suppression test. Cytotoxicity exhibited by animal interferon

Method used

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  • Recombinant pig alpha-interferon as well as preparation method and application thereof
  • Recombinant pig alpha-interferon as well as preparation method and application thereof
  • Recombinant pig alpha-interferon as well as preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0025] 1. Select the porcine alpha-interferon gene sequence

[0026] The porcine interferon alpha gene sequence with GenBank accession number 28623 was selected, and its nucleotide sequence is shown in SEQ ID NO.1. The nucleotide is translated into an amino acid sequence, as shown in SEQ ID NO.2.

[0027] 2. Optimize according to E. coli codons

[0028] The above-mentioned expressed sequence was optimized on the DNAworks online software according to the E. coli codons to eliminate conventional restriction endonuclease cleavage sites, and the RNA secondary structure was optimized. The optimized nucleotide sequence is as SEQ IDNO. As shown in 3, the last three GAA are stop codons and no protein is expressed. Among them, conventional restriction endonucleases are those well-known to those skilled in the art, such as BamHI, EcoRI, NdeI, XhoI and the like.

[0029] 3. Optimized sequence confirmation

[0030] 1) Nucleotide sequence alignment

[0031] The nucleotide sequence shown in SEQ ID ...

Embodiment 2

[0048] The embodiment of the present invention also provides a medicine containing the above-mentioned recombinant porcine alpha-interferon. Due to the high titer of the recombinant porcine interferon alpha provided by the above examples, the antiviral activity can reach 10 9 U / mg, and the content of endotoxin is extremely low, which creates prerequisites for the transformation into drugs, greatly improves the safety of drug use, and has good market potential and strong market competitiveness. The drug can be an anti-virus, anti-cell proliferation and immune-enhancing drug, and can be used alone or in combination to prevent and treat porcine viral diseases, and has potential application value.

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Abstract

The invention provides a recombinant pig alpha-interferon as well as a preparation method and application thereof and belongs to the field of genetic engineering. The recombinant pig alpha-interferonhas high antiviral activity and can be effectively applied to prevention and control of viral infectious diseases of livestock and poultry. According to the technical scheme, the preparation method comprises the following steps: optimizing a pig alpha-interferon gene, optimizing an RNA (Ribonucleic Acid) secondary structure and eliminating certain common incision enzyme sites; then cloning to a pET-23b(+) carrier to transform escherichia coli and carrying out induced expression; after carrying out isolation denaturation and purification and renaturation on an inclusion body, obtaining the recombinant pig alpha-interferon. The recombinant pig alpha-interferon provided by the invention has very high valence and the antiviral activity can reach 10<10>U / mg; the recombinant sheep tau-interferoncan be effectively applied to prevention and treatment of viral diseases of the livestock and poultry.

Description

Technical field [0001] The invention belongs to the field of genetic engineering, and particularly relates to a recombinant porcine alpha-interferon, a preparation method and application thereof. Background technique [0002] Recently, the spread of viral infectious diseases has seriously threatened the sustainable development of our country’s animal husbandry. Immunosuppressive diseases and the rapid mutation of viral antigens often lead to the failure of vaccine immunity. In addition to the continuous emergence of new viral diseases, there is still no successful vaccination prevention. On the other hand, vaccines only have a preventive effect on diseases, and treatment also depends on the use of antibiotics. The emergence of some drug-resistant strains poses a great threat to human food and health. Some countries have ordered the prohibition of certain antibiotics and antibacterial agents in aquaculture production. Therefore, there is an urgent need for a new method that is b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/21C12N1/21C07K14/56A61K38/21A61P31/12A61P35/00A61P37/04C12R1/19
CPCA61K38/00C07K14/56C12N15/70
Inventor 王雷凌红丽魏波曲信芹刘晓婧孙亚磊蒋贻海王艳玲王宏华于春梅蒋皓静武利利
Owner QINGDAO VLAND BIOTECH INC
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