Mature chicken interferon-alpha gene capable of high-efficiency expression and preparation method of polypeptide thereof

A high-efficiency expression technology for alpha interferon, which is applied in the fields of botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problem of poor renaturation effect of recombinant chicken interferon alpha and recombinant chicken interferon alpha gene Low expression level and other problems, to achieve the effect of reducing production cost, good effect, improving efficiency and yield

Inactive Publication Date: 2011-07-13
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The purpose of the present invention is to solve the problems that the existing recombinant chicken interferon alpha gene cannot be expressed in Escherichia coli or the expression level is very low, and the re

Method used

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Experimental program
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Effect test

specific Embodiment approach 1

[0017] Specific embodiment one: the sequence of the mature chicken alpha interferon gene that can be highly expressed in this embodiment is:

[0018] ATGTGCAATC ATCTGCGTCC GCAAGATGCG ACCTTTAGCC ATGATAGCCT GCAGCTGCTG

[0019] CGCGATATGG CGCCGACCCT GCCGCAGCTG TGCCCGCAGC ATAACGCGAG CTGCAGCTTT

[0020] AACGATACCA TCCTGGATAC CAGCAACACC CGCCAGGCCG ATAAACCACCATGATATC

[0021] CTGCAGCATC TGTTTAAAAT CCTGAGCAGC CCGAGCACCC CGGCGCATTG GAACGATAGC

[0022] CAGCGCCAGA GCCTGCTGAA CCGCATTCAT CGCTATACCC AGCATCTGGA ACAGTGCCTG

[0023] GATAGCAGCG ATACCCGCAG CCGCACCCGC TGGCCGCGCA ACCTGCATCT GACCATTAAA

[0024] AAACATTTTA GCTGCCTGCA TACCTTTCTG CAGGATAACG ATTATAGCGC GTGCGCGTGG

[0025] GAACATGTGC GCCTGCAGGC GCGCGCGTGG TTTCTGCATA TTCATAACCT GACCGGCAAC

[0026] ACCCGCACCTAA.

specific Embodiment approach 2

[0027] Specific embodiment two: the method for preparing the mature chicken interferon alpha polypeptide that can be highly expressed in this embodiment is realized according to the following steps: 1. Apply the online software http: / / gcua.schoedl.de / to analyze the mature chicken interferon alpha gene The rare codons in the mature chicken α-interferon gene were synonymously replaced by the E. coli preferred codons through the E. coli preferred codon table, and the optimized chicken α-interferon gene was synthesized 2. Cloning the optimized chicken interferon alpha gene into the expression vector pWL, and then transforming it into Escherichia coli competent cell Top10, inducing expression after screening, obtaining wet bacteria after centrifugation, and then carrying out inclusion body detection of wet bacteria Extraction and dissolution; 3. The dissolved inclusion bodies are renatured and purified by Sephadex G50, and the protein is collected to complete the preparation of mat...

specific Embodiment approach 3

[0033] Specific embodiment 3: The difference between this embodiment and specific embodiment 2 is that in step 2, the induced expression after screening is the culture of screening positive clone strains, and then the culture is inoculated in the culture medium containing 50 μg / ml Amp at a volume ratio of 1:100. In LB liquid medium, culture at 30°C with shaking until OD 600 0.6 to 0.8, then placed in a constant temperature water bath at 42°C to induce expression by heat shock, then continued to induce culture at 42°C for 4 hours, and centrifuged to obtain wet bacteria. Other steps and parameters are the same as in the second embodiment.

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Abstract

The invention provides a preparing method of a mature chicken interferon-alpha polypeptide, relates to a preparing method of a chicken interferon-alpha gene polypeptide, and the problems that the conventional recombinant chicken interferon-alpha gene cannot be expressed in escherichia coli, or has low expression level, and the recombinant chicken interferon-alpha has poor renaturation effect in preparation are solved. The method comprises the following steps of: 1, synonymously substituting all rare codons in the mature chicken interferon-alpha gene into escherichia coli preference codons, synthesizing to obtain optimized chicken interferon-alpha gene; 2, cloning to an expression vector, and then converting into an escherichia coli competent cell, inductively expressing after screening, centrifuging to obtain humidin, and extracting and dissolving an inclusion body; and 3, renaturing and purifying to collect protein. The mature chicken interferon-alpha gene can be expressed in high efficiency in the escherichia coli, the expression level can reach 30 percent of total mycoprotein, and the renaturation effect is good.

Description

technical field [0001] The invention relates to a preparation method of chicken alpha interferon gene and its polypeptide. Background technique [0002] For veterinary antiviral biological products, compared with traditional chemical drugs and vaccines, recombinant interferon-α has broad-spectrum and high-efficiency antiviral activity and will not cause safety problems such as drug resistance and drug residues, but recombinant chicken The alpha interferon gene cannot be expressed in Escherichia coli or the expression level is very low, and the recombinant chicken interferon alpha is difficult to refold in the preparation process, and the process is complicated, which also leads to high production costs and cannot achieve large-scale production. Contents of the invention [0003] The purpose of the present invention is to solve the problems that the existing recombinant chicken interferon alpha gene cannot be expressed in Escherichia coli or the expression level is very low...

Claims

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Application Information

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IPC IPC(8): C12N15/21C12N15/70C12R1/19
Inventor 王君伟魏双施马波高明春王巍
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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