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Human interferon-kappa mutant and preparation method thereof

A mutant and interferon technology, applied in the field of human interferon-κ mutants and its preparation, can solve the problems of free cysteine ​​correct folding and pairing interference, inactivation, etc., to achieve increased binding capacity and protein activity Effect of improving and reducing costs

Inactive Publication Date: 2021-03-19
山东晶辉生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, during the renaturation process, the existence of free cysteine ​​often interferes with the correct folding and pairing, and also makes the protein form aggregates and inactivate during the renaturation process.

Method used

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  • Human interferon-kappa mutant and preparation method thereof
  • Human interferon-kappa mutant and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1hI

[0037] Embodiment 1 hIFN-κ mutant coding gene and expression vector acquisition

[0038] The amino acid sequence of hIFN-κ is shown below:

[0039] MLDCNLLNVHLRRVTWQNLRHLSSMSNSFPVECLRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHLKQIQIGLDQQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRRRYFHRIDNFLKEKKYSDCAWEIVRVEIRR C LYYFYKFTALFRRK (SEQ ID NO: 1)

[0040] According to the hIFN-κ amino acid sequence and its high-level structure published in the prior art, it can be known that the cysteine ​​(Cys) at position 166 does not participate in the formation of disulfide bonds, so the inventors conducted site-directed mutations and constructed mutations The library is to mutate it into any of the other nineteen amino acids that do not contain a sulfhydryl group except Cys among the 20 common amino acids. The mutated sequence is as follows:

[0041] MLDCNLLNVHLRRVTWQNLRHLSSMSNSFPVECLRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHLKQIQIGLDQQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRRRYF...

Embodiment 2

[0048] Embodiment 2 protein preparation

[0049] 1. Transfer the recombinant plasmid prepared in Example 1 into Escherichia coli BL21 (DE3) competent cells, screen for kanamycin resistance, pick a single colony and carry out PCR identification, and the positive clone cells selected are those containing the recombinant plasmid pET28 -Engineering bacteria of hIFN–κ-MUT.

[0050] 2. Expression and purification of recombinant plasmid pET28-hIFN–κ-MUT in Escherichia coli BL21(DE3):

[0051] 1) Inoculate a single colony of Escherichia coli BL21(DE3) containing the recombinant plasmid pET28-hIFN-κ-MUT in 5ml of LB liquid medium containing 50μg / ml kanamycin, culture at 37°C with shaking at 200r / min for 16h, Add 5ml of bacterial liquid to 500ml of LB liquid medium containing 50μg / ml kanamycin, and expand the culture at 1:100.

[0052] 2) When the OD600 value of the Escherichia coli cultured in step 1) reaches between 0.6-0.8, add 1 mMIPTG to the bacterial solution, and culture with s...

Embodiment 3

[0056] Example 3 Activity Determination

[0057] 1. Affinity determination of interferon-κ protein binding to its receptor

[0058] The gator non-labeled biomolecular analyzer (probelife) of Suzhou Jingyuan Laboratory was used to detect the binding affinity of interferon-κ and its receptor according to the operation steps of its instruction manual: firstly, the purified interferon-κ was diluted in multiples, A total of 5 concentrations, and then let the anti-huFc probe bind to the interferon receptor first, and then react with different concentrations of interferon kappa. At this time, the receptor binds to the interferon-κ protein to form a dynamic curve, and then the reacted The probe is placed in the dissociation buffer, at this time the receptor and the interferon-κ protein will dissociate slowly to form a dynamic curve, and finally the affinity is calculated according to the curve and concentration. The specific results are shown in Table 1 and figure 1 A-B are shown. ...

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Abstract

The invention relates to a human interferon kappa (hIFNkappa) mutant and a preparation method thereof. The hIFN-kappa variant is obtained by mutating free cysteine (C) at the 166th site of an amino acid sequence shown as SEQ ID NO.1 of hIFN-kappa into serine (S) or mutating into glycine (G) or alanine (A) with a structure similar to that of serine. Compared with a strain containing a wild type hIFN-kappa sequence, the hIFN-kappa produced by expression of the plasmid strain containing the mutant sequence is relatively high in yield, higher in affinity of binding with an interferon-kappa receptor and better in in-vitro activity.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a mutant of human interferon-κ and a preparation method thereof. Background technique [0002] Interferon (Interferon, IFN) is a class of cytokines with broad-spectrum anti-virus, anti-tumor, and immunomodulatory effects. It can exert antiviral activity by binding to cognate receptor complexes on target cells. [0003] Interferon kappa (Interferon-κ, IFN-κ) is a new IFN member discovered in recent years. It shares a receptor protein with IFN-α and IFN-β, and belongs to type I interferon. IFN-κ consists of 207 amino acid residues, including a signal peptide of 27 amino acid residues at the N-terminal, and has about 30% homology with other members of type I IFN. IFN-κ is slightly larger than other type I IFNs, There is an insertion of 12 amino acid residues between the C and D helices. The IFN-κ gene is located on the short arm of the ninth pair of chromosomes...

Claims

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Application Information

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IPC IPC(8): C07K14/555C12N15/20C12N15/70C12N1/21A61K38/21A61P35/00A61P31/12C12R1/19
CPCC07K14/555C12N15/70A61P35/00A61P31/12A61K38/00
Inventor 彭继先钱文正柴辉闫韵秋戴文宇李振森于海勤
Owner 山东晶辉生物技术有限公司
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