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Preparation method of canine interferon alpha 2 recombinant protein

A technology of canine interferon and recombinant protein, which is applied in the field of preparation of canine interferon-α2 recombinant protein, can solve the problems such as the expression of canine interferon-α2 recombinant protein, etc., and achieves the effect of improving activity

Pending Publication Date: 2018-08-21
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the expression of canine interferon-α2 recombinant protein using the E. coli expression system

Method used

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  • Preparation method of canine interferon alpha 2 recombinant protein
  • Preparation method of canine interferon alpha 2 recombinant protein
  • Preparation method of canine interferon alpha 2 recombinant protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Design and Synthesis of Gene Sequence and Primers

[0039] Referring to the CaIFN-α2 gene sequence (M28625.1) in Genbank, codon optimization was performed according to the codon preference of the Escherichia coli expression system. The optimized gene was synthesized by Nanjing GenScript Biotechnology Co., Ltd. BamH I and Xho I restriction sites were added to the ' and 3' ends to obtain the CaIFN-α2 gene fragment. See SEQ ID NO:1 for the sequence. Using the synthetic CaIFN-α2 gene sequence as a template, the signal peptide was excised, and only the coding gene of the mature peptide (SEQ ID NO: 2) was retained, and a pair of specific primers were designed using Primer 5 software, among which the upstream primer CaIFN-α2-F The Nde I restriction enzyme cutting site was introduced, and the BamH I restriction enzyme cutting site was introduced into the downstream primer CaIFN-α2-R. Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequence o...

Embodiment 2

[0040] Example 2 Construction of pET-32a-CaIFN-α2 recombinant expression plasmid

[0041] The pET-32a vector and the optimally synthesized CaIFN-α2 gene fragment were double-digested with BamH I and Xho I, respectively, and the digested expression vector fragment and target fragment were gel-recovered. T4 ligase was used to connect the CaIFN-α gene fragment and the pET-32a expression vector to construct the pET-32a-CaIFN-α2 recombinant expression plasmid. The bands of 5900bp and 528bp are consistent with the size of the expression vector pET-32a and the target gene band of CaIFN-α2, such as figure 1 shown. PCR identification was carried out on the selected plasmid, and a 528bp band was amplified, which was consistent with the theoretical size, see figure 2 . It indicated that the recombinant plasmid pET-32a-CaIFN-α2 was constructed successfully. The positive plasmid was sent to Jinweizhi Company for sequencing.

Embodiment 3

[0042] Example 3 Construction of pET-21a-CaIFN-α2 recombinant expression plasmid

[0043] Using the pET-32a-CaIFN-α2 recombinant expression plasmid as a template and CaIFN-α2-F / CaIFN-α2-R as primers, the CaIFN-α2 mature peptide gene fragment was amplified by polymerase chain reaction (PCR). 25 μL PCR system: PreMix 12.5 μL, upstream and downstream primers 1 μL, template 1 μL, double distilled water to make up to 25 μL. The PCR reaction conditions were as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 15 s, annealing at 62°C for 15 s, extension at 72°C for 30 s, and 30 cycles; final extension at 72°C for 10 min. After PCR was completed, the product was subjected to 1% agarose gel electrophoresis ( image 3 ), according to the DNA recovery kit instructions for recovery and purification.

[0044] The pET-21a vector was digested with Nde I / BamH I, and recovered and purified according to the instructions of the DNA recovery kit. Add 1 μL of linearized pET-...

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Abstract

The invention provides a preparation method of canine interferon alpha 2 recombinant protein. The preparation method comprises following steps: canine interferon alpha 2 gene is subjected to codon optimization, and cloning into pET-21a or pET-32a for construction of recombinant expression plasmids is carried out, transformation into competent escherichia coli cells BL21 is carried out, preparationof recombinant bacteria is carried out, and then fermentation, inducible expression, and purifying are carried out so as to obtain the canine interferon alpha 2 recombinant protein. The canine interferon alpha 2 recombinant protein can be taken as a wide spectrum antiviral preparation, and possesses obvious preventing and controlling effect on infection of canine distemper virus, canine parvovirus disease, canine parainfluenza virus, and infectious canine hepatitis virus. The preparation method is capable of providing an effective technical mean for realization of recombinant canine interferon alpha 2 technological production and further broad spectrum prevention of canine virus.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biological products, in particular to a preparation method of canine interferon-α2 recombinant protein. Background technique [0002] Interferon (IFN) is an active protein with multiple functions, produced by monocytes and lymphocytes, and is one of the most studied and widely used cytokines. It is a glycoprotein secreted by recipient cells when the body cells are stimulated by viruses, bacterial endotoxins and other inducers. Interferon can be divided into type I, type II, and type III according to the cell, receptor, activity, etc. in which interferon is produced. IFN-α belongs to type I interferon and has many subtypes. IFN-α has broad-spectrum antiviral activity, which can not only inhibit the replication of RNA viruses, but also inhibit the replication of various DNA viruses. Its antiviral activity is mainly manifested in the following aspects: ①It can inhibit infection by promoting ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/21C12N15/70C12N15/66C07K14/56A61K38/21A61P31/12A61P31/14A61P31/20
CPCA61K38/00A61P31/12A61P31/14A61P31/20C07K14/56C12N15/66C12N15/70
Inventor 贾红朱鸿飞宋天琪鑫婷侯绍华姜一曈
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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