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Canine recombinant interferon alpha7, and preparation method and application thereof

A technology of recombinant interferon and recombinant plasmid, which is applied in the fields of genetic engineering and biological products, can solve the problems of natural interferon's spatial conformation gap and low immunogenicity, and achieve the effect of increasing production

Active Publication Date: 2020-04-14
ANHUI JIUCHUAN BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, because the recombinant interferon expressed in prokaryotes cannot undergo post-translational modification, there is a certain gap between the spatial conformation and the natural interferon, and its immunogenicity is low

Method used

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  • Canine recombinant interferon alpha7, and preparation method and application thereof
  • Canine recombinant interferon alpha7, and preparation method and application thereof
  • Canine recombinant interferon alpha7, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Optimization of gene sequence encoding canine interferon-α7 and design and synthesis of primers

[0032] According to the cDNA sequence of canine interferon-α7 in GenBank (NM_001006654.1) as shown in SEQ ID No.2, the present invention optimizes codons and synthesizes the target gene sequence. The codons of the gene encoding canine interferon-α7 used the most preferred codons of Pichia pastoris. The optimized gene sequence of canine interferon-α7 is shown in SEQ ID No.3, and the amino acid sequence of its encoded protein is shown in SEQ ID No.1.

Embodiment 2

[0033] Example 2 Construction of pPICZαA-CaIFN-α7 expression vector

[0034] The CaIFN-α7 gene fragment shown in Example 1 as shown in SEQ ID No.3 and the vector pPICZαA were double-digested with EcoRI and NotI, and the double-digested fragment was recovered. The ratio of the CaIFN-α7 gene fragment and the vector pPICZαA was 3:1 The molar ratio was connected overnight at 16°C, transformed into Escherichia coli competent cells DH5a, plated on a low-salt LB plate containing Zeocin (25ug / mL), and incubated 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 pPICZαA-CaIFN-α7 vector is shown in figure 1 .

Embodiment 3

[0035] Example 3 Electroporation Transformation of Yeast Cells and Screening of High Expression Transformants

[0036] Pick the colonies that have been identified and sequenced correctly by PCR for expansion culture, select the high-purity plasmid extraction kit from Tiangen Biochemical Reagent Co., Ltd., and perform plasmid extraction according to the instructions. Prepare Pichia X33 competent cells according to the instructions of Invitrogen Pichia Expression Kit. The recombinant plasmid was linearized with restriction endonuclease SacI, and digested in a metal bath at 37°C for 5h. See Table 1 for the linearization system.

[0037] Table 1

[0038]

[0039]

[0040] The linearized plasmid was then recovered using ethanol precipitation. Add 0.1 volume of 3mol / L NaAc (PH5.2) and 2.5 volumes of absolute ethanol to the linearized plasmid, place at -20°C for more than 30min, centrifuge at 14,000r / min for 20min at 4°C, and discard the supernatant. Add 700 μL of 75% ethanol...

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Abstract

The invention discloses a canine recombinant interferon alpha7, and a preparation method and an application thereof. The nucleotide sequence of the canine recombinant interferon alpha7 is obtained through codon optimization, a Pichia pastoris strain expression system is used for expressing recombinant yeast engineering bacteria containing the canine recombinant interferon alpha7 gene with the optimized codon, and the secreted and expressed high-activity canine recombinant interferon alpha7 is obtained. The canine recombinant interferon alpha7 prepared by the method is high in purity, the protein concentration after purification reaches 0.55 mg / ml, the protein yield of 1 L of recombinant yeast engineering bacteria reaches 150 mg, the antiviral specific activity of the recombinant protein onVSV on MDCK cells can reach 1.85 * 10<6> IU / mg, studies on the expression, fermentation and activity of CaIFN-alpha7 in an eukaryotic yeast system are of great significance in early industrial production of canine interferon alpha.

Description

technical field [0001] The invention belongs to the field of genetic engineering and biological products, and in particular relates to a canine recombinant interferon α7 and its preparation method and application. Background technique [0002] Interferon is a class of important substances that have comprehensive antiviral effects, enhance specific and nonspecific immune responses, and can regulate the growth process of normal cells and tumor cells. Since the discovery of interferon, people have found the existence of interferon in various mammals, fish, insects and other animals, confirming the widespread existence of interferon. According to the gene homology, structural characteristics, receptors and biological activities of different interferons, they are divided into three types, namely type I, type II and type III. Type Ⅰ interferon includes IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-τ and IFN-ω, type Ⅱ only includes γ interferon, and IFN-λ is Ⅲ discovered in 2003 type in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/56C12N15/21C12N15/81C12N1/19A61K38/21A61P31/14C12R1/84
CPCA61K38/00A61P31/14C07K14/56C12N15/815
Inventor 王红朵王梦夏兵兵丁爽李诚茹吴蕾凡玉芳王亚男吴博张勇
Owner ANHUI JIUCHUAN BIOTECH
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