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Canine recombinant interferon-lambda 1, and preparation method and application thereof

A technology for recombining interferon and amino acids, which is applied in the directions of interferon, botanical equipment and methods, biochemical equipment and methods, etc., to achieve high safety and good application prospects.

Inactive Publication Date: 2018-08-03
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

Traditional treatment programs are difficult to be effective clinically, so active treatment and prevention of viral diseases in dogs is the most concerned issue in the industry

Method used

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  • Canine recombinant interferon-lambda 1, and preparation method and application thereof
  • Canine recombinant interferon-lambda 1, and preparation method and application thereof
  • Canine recombinant interferon-lambda 1, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Optimization of gene sequence encoding canine interferon-λ1 and design and synthesis of primers

[0040] According to the cDNA sequence of canine interferon-λ1 in GenBank (No. AB819731.1), the present invention optimizes the codon and synthesizes the target gene sequence. The codons of the gene encoding canine interferon-λ1 used the most preferred codons of Pichia pastoris. In order to prevent the GC content of the translated mRNA from being too high and the secondary structure of the mRNA from affecting the translation efficiency, the present invention uses a sub-favored code for certain amino acids, provided that the frequency of use of the sub-favored code is very close to that of the most preferred code, and the amino acid sequence The original sequence remains unchanged. In some very special cases, in order to reduce or increase restriction sites, the sequence at certain positions is properly adjusted. Therefore, the present invention optimally designed ...

Embodiment 2

[0041] Example 2 Construction of pPICZαA-CaIFN-λ1 vector

[0042] The CaIFN-λ1 gene fragment and the carrier pPICZαA in Example 1 were double-digested with XhoI and NotI, and the double-digested fragment was recovered. The CaIFN-λ1 gene fragment and the carrier pPICZαA were ligated overnight at 4°C at a molar ratio of 3:1, and transformed into Escherichia coli competent cells DH5α were plated on a low-salt LB plate containing Zeocin (25ug / ml), and cultured at 37°C for 16-24h. Pick a single colony, extract the plasmid, perform PCR identification and send it to Sangon for sequencing. The schematic diagram of the construction of the pPICZαA-CaIFN-λ1 vector is shown in figure 1 .

Embodiment 3

[0043] Example 3 Electroporation Transformation of Yeast Cells and Screening of High Expression Transformants

[0044] Pick colonies with correct PCR and sequencing for expansion culture, select Beyond’s Plasmid Midi Preparation Kit, and carry out plasmid extraction according to the instructions. Prepare Pichia X-33 competent cells according to the Invitrogen PichiaExpression Kit operating instructions. The pPICZαA-CaIFN-λ1 plasmid was linearized using Sac I. See Table 1 for the linearization system.

[0045] Table 1

[0046]

[0047] 37°C overnight. The linearized pPICZαA-CaIFN-λ1 plasmid was subjected to gel electrophoresis, and the size of the result was consistent with the expectation. See the results in figure 2 .

[0048] Mix 200 μl competent cells with 20 μl (10-15 μg) SacI-linearized recombinant plasmid pPICZαA-CaIFN-λ1, inject it into a pre-cooled 0.2 cm electrode cup, place it on ice for several minutes, and place it on the Bio-Rad Gene Pulser electrotransfe...

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Abstract

The invention provides a canine recombinant interferon-lambda 1, and a preparation method and an application thereof. The canine recombinant interferon-lambda 1 has a nucleotide sequence as shown in SEQ ID NO. 1. The invention also provides a preparation method for the canine recombinant interferon-lambda 1. According to the invention, a pichia pastoris strain expression system is utilized to express a recombinant yeast engineering strain containing a codon-optimized canine recombinant interferon-lambda 1 gene, so secretion-expressed canine recombinant interferon-lambda 1 with high activity isobtained. The canine recombinant interferon-lambda 1 prepared by using the method provided by the invention has high purity, can reach a purity of 0.86 mg / ml after purification, has a protein yield of 42 mg in 1 L of recombinant yeast engineering strains, has a specific antiviral activity of 2.85 x 10<7> IU / mg, can effectively prevent and treat infectious diseases like canine distemper, canine parvovirus, canine parainfluenza virus infection and canine infectious hepatitis, and has high safety and good application prospects.

Description

technical field [0001] The invention belongs to the field of biological products, discloses a highly active canine recombinant interferon-λ1, and also provides a preparation method and application for highly expressing canine recombinant interferon-λ1. Background technique [0002] In recent years, my country's pet industry has developed rapidly, and the number of pet dogs and cats is increasing day by day. Pet-derived viral diseases are currently a serious disease. The most common canine distemper and canine parvovirus are usually serious hazards, with high morbidity, strong infectivity, and high mortality. They are the most serious infectious diseases that endanger the dog industry in my country and seriously restrict the development of the pet industry. Traditional treatment programs are difficult to be effective clinically, so active treatment and prevention of viral diseases in dogs is the most concerned issue in the industry. [0003] According to the difference of am...

Claims

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Application Information

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IPC IPC(8): C07K14/555C12N15/20C12N15/81C12N1/19A61K38/21A61P31/14A61P31/20C12R1/84
CPCA61K38/00A61P31/14A61P31/20C07K14/555C12N15/815
Inventor 贾红侯绍华郭晓宇鑫婷朱鸿飞姜一曈陈美荣
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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