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Low-saccharification mutant interferon lambda1 as well as expression and purification methods and application thereof

A mutant and interferon technology, applied in the direction of interferon, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of increasing immunogenicity and changing glycoproteins, and achieve good uniformity, improved uniformity, Apply promising effects

Active Publication Date: 2012-02-15
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

This non-human glycosylation modification of some glycoproteins may increase immunogenicity, bind to mannose receptors on the surface of human cell membranes to generate an immune response, and some may also change the functions and characteristics of glycoproteins, limiting the ability of Pichia Therapeutic applications of recombinant glycoproteins expressed by yeast [Hamilton S.R., Bobrowicz P., Bobrowicz B., Davidson R.C., Li H., Mitchell T., Nett J.H., Rausch S., Stadheim T.A., Wischnewski H., Wildt S. ., Gerngross T.U. (2003) Production of Complex Human Glycoproteins in Yeast. Science, 301: 1244-46. Daly R. and Hearn M.T.W. (2005). Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production .J.Mol.Recognit.18:119-138]

Method used

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  • Low-saccharification mutant interferon lambda1 as well as expression and purification methods and application thereof
  • Low-saccharification mutant interferon lambda1 as well as expression and purification methods and application thereof
  • Low-saccharification mutant interferon lambda1 as well as expression and purification methods and application thereof

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Embodiment 1

[0033] Embodiment 1, the acquisition of low glycation mutant interferon-λ1 gene (IFN-λ1-Nm) of the present invention

[0034]The N-glycosylation site of the IFN-λ1 gene fragment was subjected to site-directed mutation using overlap extension PCR technology, and the codon sequence aac of Asn in the IFN-λ1 gene fragment was replaced with the codon sequence caa of Gln, so that the IFN-λ1 46NWS48 in the amino acid sequence was changed to 46QWS48, and the primer sequence used was: IFNλ1-Nm-F:

[0035] 5’-CAAGCTGAAA TGGAGTTGCA-3' and IFNλ1-Nm-R:5'-TGCAACTCCA TTTCAGCTTG-3', lowercase italics and black bases are codons for Gln.

[0036] pA-αF-IFNλ1 20 The plasmid is used as a template (the construction process of the plasmid is described in detail in the invention patent of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, patent No. ZL 200510115720.3), and the first round of PCR uses two pairs of primers to amplify the two target fragments. Increment...

Embodiment 2

[0037] Example 2, Construction of IFN-λ1-Nm Special Expression Vector pA-6IFNλ1-Nm

[0038] Plasmid pUC-IFN-λ1-Nm was digested with restriction endonuclease EcoR I, and an insert fragment of about 818 bp was recovered, ligated with pAO815 plasmid vector (purchased from Invitrogen Company) linearized by EcoR I, transformed into Escherichia coli DH5α, and selected positive Cloning, plasmid extraction, recombinant plasmid pA-IFNλ1-Nm was obtained, the plasmid was verified by restriction enzyme digestion with EcoR I and PstI, and then sequencing verification was carried out. The verification results showed that the insertion position, direction and sequence of the IFN-λ1-Nm gene were obtained ( Consistent with the nucleotide sequence of SEQ ID No.2 in the sequence listing) all correct recombinant methanolophilic yeast expression vectors, the expression vectors contain IFN-λ1-Nm gene expression unit, namely from upstream to downstream including alcohol oxidase (AOX ) promoter, the ...

Embodiment 3

[0040] Example 3. Transformation, expression and purification of IFN-λ1-Nm.

[0041] 1. Transformation of methanolophilic yeast

[0042] Take 10 μg of plasmid pA-6IFNλ1-Nm, digest it with restriction endonuclease Sal I, transform the methanolophilic yeast strain GS115 with the electroporation transformation method, and spread the transformed bacteria on MD medium (1.34% YNB, 4×10 5 % biotin, 2% glucose) at 30°C for 4-6 days, screened to obtain a recombinant yeast strain for expressing the low glycation mutant interferon-λ1 (IFN-λ1-Nm), named GS115 / pA-6IFNλ1- Nm.

[0043] 2. Expression of low glycation mutant interferon-λ1 (IFN-λ1-Nm)

[0044] Inoculate the monoclonal GS115 / pA-6IFNλ1-Nm recombinant yeast strain in 10mL BMG medium (100mM potassium phosphate, pH6.0, 1.34%YNB, 4×10 5 % biotin, 10% glycerol), shake culture at 30°C for 24h, transfer to 100mL BMG medium for 24h, centrifuge to collect the bacteria, resuspend in 500mL BMMY medium (100mM potassium phosphate, pH6.0, 1...

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Abstract

The invention discloses low-saccharification mutant interferon lambda1 as well as expression and purification methods thereof. The low-saccharification mutant interferon lambda1 is protein obtained after replacing Asn in the N46 bit in the IFN-lambda1 mature peptide sequence into Gln. The blockage on the high-mannose modification process of the N-glycosylation site of the IFN-lambda1 by the Pichia pastoris is realized so that the IFN-lambda1-Nm genes are correctly expressed in the Pichia pastoris in a high-efficiency secretory way, the uniformity of the expressed recombination IFN-lambda1-Nm protein is obviously improved, the expression level is higher than 65mg / L, the purity of the expressed recombination protein after two-step column chromatography is higher than 98 percent, and the low-saccharification modified recombination IFN-lambda1-Nm protein has the same biological activity as the original Pichia pastoris expressed wild IFN-lambda1 and simultaneously has better safety. The invention provides an effective method for efficient low-cost mass production of low-saccharification mutant interferon lambda1 with biological activity and good uniformity, higher practical application values are realized, and the application prospects are wide.

Description

technical field [0001] The invention belongs to the protein and its expression and purification method in the field of biotechnology, in particular to a biologically active low-glycosylation mutant interferon-λ1 (IFN-λ1) and its expression and purification method and anti-tumor cell proliferation role. Background technique [0002] IFN-λs is a newly discovered class of interferon-like cytokines with antiviral activity, including IFN-λ1 (IL-29), IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B). IFN-λs is very similar to IL-10 family in gene structure, and closer to type I IFN in amino acid composition and function, establishing an evolutionary link between IL-10 family and type I IFN. IFN-λs, especially IFN-λ1, has the potential value of being developed into broad-spectrum antiviral drugs, anti-tumor drugs and immune-enhancing drugs. [0003] Interferon (interferon, IFN) is the earliest cytokine recognized by humans. As early as 1957, Isaacs and Lindenmann discovered a protein with anti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/555C12N15/20C12N15/81C12N1/19C07K1/18C07K1/16A61K38/21A61P35/00C12R1/84
Inventor 陈虹黄秉仁惠希武谢云飞陈等马晓骊王欣
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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