High-activity porcine interferon alpha mutant with 7 mutation loci and preparing method and application thereof

A porcine interferon and site mutation technology, applied in the field of interferon, can solve unresolvable problems and achieve the effect of reducing misfolding, high cost, and difficult cell lines

Inactive Publication Date: 2019-10-18
ANYANG INST OF TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Chen Tao et al mutated the 86th cysteine ​​C to tyrosine Y with reference to the transformation method of human interferon α, and obtained a protein activity of 5

Method used

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  • High-activity porcine interferon alpha mutant with 7 mutation loci and preparing method and application thereof
  • High-activity porcine interferon alpha mutant with 7 mutation loci and preparing method and application thereof
  • High-activity porcine interferon alpha mutant with 7 mutation loci and preparing method and application thereof

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Embodiment 1

[0037] 1. Obtain the target gene

[0038]Design primers: According to the mature nucleotide sequence of porcine interferon-alpha contained in the GenBank database (accession number: AB369102.1), design two pairs of primers to amplify porcine interferon-alpha, F-1 and R-1 amplify the complete Porcine alpha interferon, F-86, R-86, R-156, F-38, R-38 contain mutation sites. The primers were synthesized by Zhengzhou Shangya Biological Engineering Co., Ltd.: the sequence of primer F-1 is shown in SEQ ID NO.3, the sequence of R-1 is shown in SEQ ID NO.4, and the sequence of F-86 is shown in SEQ ID NO.5 The R-86 sequence is shown in SEQ ID NO.6, the R-156 sequence is shown in SEQ ID NO.7, the F-38 sequence is shown in SEQ ID NO.8, and the R-38 sequence is shown in SEQ ID NO. 9.

[0039] Target gene fragment amplification and electrophoresis identification: use the pMD18T-IFN-α plasmid stored in the laboratory (by using the method of preserving the target gene, the obtained target fr...

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Abstract

The invention belongs to the field of interferons, and particularly relates to a high-activity porcine interferon alpha mutant with 7 mutation loci and a preparing method and application thereof. According to the high-activity porcine interferon alpha mutant with the 7 mutation loci and the preparing method and application thereof, a porcine interferon alpha gene is modified to obtain a new gene sequence, and through modification, the biological activity of the interferon is improved. An overlapped PCR method is applied to conducting site-specific mutation on the natural porcine interferon alpha gene, the modified gene is cloned to a pET-32a carrier to construct a recombinant plasmid, then the recombinant plasmid which is accurately identified is cloned into Rosetta expression bacteria forinduction expression and identification, recombinant protein is expressed in the form of an inclusion body, the molecular mass of the protein is about 32 KDa, after purification and dialysis treatment, the porcine interferon alpha mutant with high purity is obtained, and the antiviral activity is determined through cytopathic effect inhibition. As is shown through results, the antiviral activityof the mutant protein obtained through experiments is 8.15*10<8> U/mg, and increased by about 200 times compared with that of wild-type interferons expressed in vitro.

Description

technical field [0001] The invention belongs to the field of interferon, and relates to the preparation of mutant interferon, in particular to a highly active 7-site mutated porcine interferon alpha mutant and its preparation method and application. Background technique [0002] Porcine Interferon (PoIFN) has a wide range of good defense and inhibitory effects on most viral swine diseases, and is safe and reliable. It is regarded as the most promising "penicillin" for the treatment of viruses. Porcine leukocyte interferon is widely used in the market. It is a natural porcine alpha interferon. Its main advantages are: it has significant antiviral and immune regulation functions; it can stimulate the antiviral activity of animals in a relatively short period of time. Function; the antiviral spectrum is relatively broad, and can resist the mixed infection of several viruses at the same time; green environmental protection, low drug residue, no toxic side effects, no withdrawal ...

Claims

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Application Information

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IPC IPC(8): C07K14/56C12N15/21C12N15/70C07K1/36C07K1/34C07K1/30A61K38/21A61P31/00
CPCC07K14/56C12N15/70A61P31/00A61K38/00
Inventor 梁秀丽魏法山魏战勇郑兰兰王朋涛夏璐张福良王雪平龙福庆贾雪娜
Owner ANYANG INST OF TECH
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