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Preparation method of fusion protein of porcine interferon-alpha 1 and thymosin-alpha 1

A porcine interferon, fusion protein technology, applied in recombinant DNA technology, peptides, hybrid peptides and other directions, can solve the problem of single therapeutic drug, achieve simple preparation process, protect cells from virus attack activity, and strong operability Effect

Inactive Publication Date: 2014-07-23
南京洲邦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In view of this increasingly serious problem, there is an urgent need to produce a drug that is specific to pig diseases and has high biological activity and broad-spectrum anti-virus. At present, hyperimmune serum is mainly used in the clinical treatment of viral diseases, and the treatment drug is relatively single

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] In the process of preparing the fusion protein of porcine interferon α1 and thymosin α1 in this example, the concentration of IPTG to induce the expression of the fusion protein was 0.5 mmol / L.

[0017] 1. Primer Design

[0018] The mature peptide gene sequence of porcine IFNα1 (sequence number DQ249000) and the protein sequence of THYα1 (sequence number 770552A) were found in GenBank. After codon preference optimization, SOE-PCR primers were designed using Primer Premier 5.0 software based on the optimized gene sequence , the primers are:

[0019] Primer 1: 5'-CATATGTGCGATCTGCCGCAGACCC-3';

[0020] Primer 2: 5'-CGAGCCGCCGCCGCCGCCGCGCGCCGAGCCGCCGCCGCCCAGATTACGGCTGCTGCTAAACGC-3';

[0021] Primer 3: 5'-GGCGGCGGCGGCTCGGCGCGCGGCGGCGGCGGCGGCTCGTCGGATGCGGCGGTGGAT-3';

[0022] Primer 4: 5'-CTCGAGTCATTAATTTTCCGCTTCTTCC-3'.

[0023] At the same time, Nde I and Xho I restriction sites were introduced into primer 1 and primer 4, respectively.

[0024] 2. Construction of porci...

Embodiment 2

[0035] In the process of preparing the fusion protein of porcine interferon α1 and thymosin α1 in this example, the concentration of IPTG to induce the expression of the fusion protein was 1 mmol / L. The preparation technique of the fusion protein of porcine interferon α1 and thymosin α1 is the same as that in Example 1.

[0036] Results The target protein of IFNα1-THYα1 was purified, and the content of the target protein was determined by Beyontian BCA protein detection kit, and the concentration of porcine IFNα1-THYα1 fusion protein was about 120 mg / L. The collected porcine IFNα1-THYα1 fusion protein eluate was washed with PEG (Mr 6000) and shrunk to the required volume, added with 0.02% NaN3 preservative, and stored at 4°C for later use.

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Abstract

The invention belongs to the field of veterinary biological pharmacy, and discloses a preparation method of a fusion protein of porcine interferon-alpha 1 and thymosin-alpha 1. The preparation method comprises following steps: an amplification primer is synthesized based on porcine interferon-alpha 1 (sequence number DQ249000) gene sequence and thymosin-alpha 1(sequence number 770552A) sequence which are disclosed by GenBank; fusion amplification of complete genome sequences of porcine interferon-alpha 1 and thymosin-alpha 1 is carried out; cloning and inducible expression of glutathione-s-transferase-interferon alpha 1-thymosin alpha 1 fusion protein are carried out; detection and identification are carried out; and the fusion protein is separated and purified. The fusion protein is capable of maintaining respective biological functions of porcine interferon-alpha 1 and thymosin-alpha 1 preferably, possesses certain activity in protecting cells from virus attack and promoting cell surface receptor expression, and is capable of providing industrialized production of porcine interferon-alpha 1 and thymosin-alpha 1 fusion protein preparations with technological support.

Description

technical field [0001] The invention relates to the field of veterinary biopharmaceuticals, in particular to a preparation technology for fusion protein of porcine interferon α1 and thymosin α1. Background technique [0002] Interferon (IFN) is a small peptide secreted by cells, which has a wide range of biological activities such as anti-virus, anti-proliferation and immune regulation. Porcine interferon (poIFN), which is similar to human interferon (HuIFN), is also divided into type I and type II. Porcine interferon α1 (poIFNα1) is a type I interferon, which has broad-spectrum, high-efficiency anti-virus functions, and has the characteristics of small toxic and side effects, quick-acting and multipotent, and plays a key role in regulating the immune system. Species specificity is one of the basic characteristics of interferon, but porcine leukocyte interferon (PLeIFN, whose main component is alpha interferon) can show strong antiviral activity on many heterogeneous cells....

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K19/00
Inventor 王占伟孙玉贤武昱孜
Owner 南京洲邦生物科技有限公司
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