阅读说明：本技术 干扰素-κ突变体及其制备方法 (Interferon-kappa mutant and preparation method thereof ) 是由 赵耀 张建军 魏婷婷 孟万利 张秋磊 于 2021-04-21 设计创作，主要内容包括：本发明公开了一种干扰素-κ突变体,其通过在野生型干扰素-κ的基础上,将自由的半胱氨酸残基突变为半胱氨酸之外的氨基酸,和/或将能够形成氧化修饰的甲硫氨酸残基部分或全部突变为甲硫氨酸之外的氨基酸。本发明的干扰素-κ突变体体外复性和纯化方便,并且产物均一。(The invention discloses an interferon-kappa mutant, which is obtained by mutating free cysteine residues into amino acids except cysteine and/or mutating partial or all methionine residues capable of forming oxidation modification into amino acids except methionine on the basis of wild-type interferon-kappa. The interferon-kappa mutant of the invention has convenient in-vitro renaturation and purification and uniform product.)

1. An interferon-kappa mutant characterized in that it is obtained by mutating a free cysteine residue to an amino acid other than cysteine and/or by mutating a methionine residue capable of forming an oxidative modification partially or entirely to an amino acid other than methionine on the basis of a wild-type interferon-kappa.

2. The interferon- κ mutant of claim 1, wherein the free cysteine residue is mutated to an amino acid with a smaller side chain; and (2) partially or completely mutating the methionine residue capable of forming the oxidation modification into one or more of amino acid with smaller side chain and/or hydrophobic amino acid.

3. The interferon- κ mutant of claim 2, wherein the side chain-smaller amino acid is serine, alanine or glycine; the hydrophobic amino acid is valine, leucine or isoleucine.

4. The interferon-kappa mutant according to claim 1, wherein the interferon-kappa mutant further has one or more amino acids added to the N-terminus based on a wild-type interferon-kappa.

5. The interferon-kappa mutant of claim 4, wherein the one or more amino acids added at the N-terminus are amino acids having a smaller side chain.

6. The interferon-kappa mutant of claim 5, wherein the amino acid having a smaller side chain is serine, alanine, or glycine.

7. The interferon- κ mutant according to claim 1, wherein the amino acid sequence of the wild-type interferon- κ is as shown in SEQ ID No.1, or comprises the amino acid sequence as shown in SEQ ID No.1, or comprises an amino acid sequence at least 80%, 90%, 95%, 98%, 99% identical to SEQ ID No. 1.

8. The interferon-kappa mutant of claim 7, wherein the free cysteine residue is cysteine residue 166 of the sequence shown in SEQ ID No. 1; the methionine residues capable of forming oxidation modification are methionine residues at position 52, 112, 115 and 120 of the sequence shown in SEQ ID NO. 1.

9. The interferon- κ mutant of claim 8, wherein the amino acid sequence of the interferon- κ mutant is as follows:

1) on the basis of the amino acid sequence shown in SEQ ID NO.1, the 166 th cysteine residue is replaced by serine, alanine or glycine;

2) on the basis of the amino acid sequence shown in SEQ ID NO.1, the 166 th cysteine residue is replaced by serine, alanine or glycine, and serine, alanine or glycine is added at the N terminal;

3) on the basis of the amino acid sequence shown in SEQ ID No.1, the 166 th cysteine residue is replaced by serine, alanine or glycine, the N-terminal is added with serine, alanine or glycine, and the 112 th methionine residue and the 115 th methionine residue are replaced by serine, alanine, glycine or hydrophobic amino acid;

4) on the basis of the amino acid sequence shown in SEQ ID NO.1, the 166 th cysteine residue is replaced by serine, alanine or glycine, the N-terminal is added with serine, alanine or glycine, and the 115 th methionine residue is replaced by serine, alanine, glycine or hydrophobic amino acid;

5) on the basis of the amino acid sequence shown in SEQ ID No.1, the 166 th cysteine residue is replaced by serine, alanine or glycine, the N-terminal is added with serine, alanine or glycine, and the 115 th methionine residue and the 120 th methionine residue are replaced by serine, alanine, glycine or hydrophobic amino acid;

6) on the basis of the amino acid sequence shown in SEQ ID No.1, the 166 th cysteine residue is replaced by serine, alanine or glycine, the N-terminal is added with serine, alanine or glycine, and the 52 nd methionine residue, the 115 th methionine residue and the 120 th methionine residue are replaced by serine, alanine, glycine or hydrophobic amino acid;

7) comprising an amino acid sequence as set forth in any one of 1) to 6); or

8) Comprising an amino acid sequence which is at least 80%, 90%, 95%, 98%, 99% identical to the amino acid sequence shown in any one of 1) to 6).

10. An isolated polynucleotide encoding the interferon- κ mutant according to any one of claims 1-9.

11. The isolated polynucleotide of claim 10, wherein the polynucleotide has a sequence as set forth in SEQ ID No.4 or 6, or comprises a polynucleotide sequence that is at least 80%, 90%, 95%, 98%, 99% identical to a polynucleotide sequence of SEQ ID No.4 or 6.

12. An expression vector comprising an isolated polynucleotide of claim 10 or 11.

13. A host cell comprising an isolated polynucleotide of claim 10 or 11 or an expression vector of claim 12.

14. A method for producing an interferon- κ mutant according to any one of claims 1 to 9; the preparation method comprises the following steps: expressing a polynucleotide sequence encoding an interferon-kappa mutant according to any one of claims 1 to 9 in an expression system or using an expression vector according to claim 12 or by a host cell according to claim 13.

15. The method of claim 14, wherein the expression system is a prokaryotic expression system.

16. The method for preparing interferon-kappa mutants according to claim 15, wherein the polynucleotide sequence is ligated to an expression vector pET21b or pET41a and expressed in e.coli BL21(DE 3).

17. The method of making an interferon- κ mutant according to claim 16, further comprising: the inclusion body of interferon-kappa mutant obtained by expression is subjected to in vitro renaturation and purification.

18. The method of claim 17, wherein the in vitro renaturation is performed by inclusion body solubilization using guanidine hydrochloride and/or DTT and renaturation in a renaturation solution; the purification is ion exchange chromatography purification and/or reverse-phase chromatography purification.

19. Use of an interferon-kappa mutant for the manufacture of a medicament or formulation for the treatment of a disease, wherein the interferon-kappa mutant is as defined in any one of claims 1 to 9 or is obtainable by the manufacturing process as defined in any one of claims 14 to 18.

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to an interferon-kappa mutant and a preparation method thereof.

Background

Interferon (IFN), an anti-influenza virus factor that is secreted by chicken embryos infected with influenza virus and discovered by scientists Isaacs and Lindenmann in 1957 (Isaacs et al, 1957), is a protein with wide biological activity, is an important cytokine that can be synthesized and secreted by a variety of cells, has various effects of resisting virus, resisting tumor, regulating immunity and the like (Takaoka et al, 2003), and is an important component of a natural immune defense system of an organism.

Interferons belong to induced proteins, normal cells generally do not spontaneously generate, and the interferons belong to an immune system which plays the fastest virus defense function. The antiviral effect of interferon is usually earlier than the specific immune response of the organism, can effectively limit the virus replication, has the activities of inhibiting the proliferation of tumor cells and regulating the immune function (LaFleur et al, 2001; Nardelli et al, 2002), and is a medicine which is licensed in the world and is used for treating various viral diseases, tumors and immune disorders. The existing antiviral drugs only have a simple antiviral effect, and IFN has double effects of resisting viruses and regulating immunity, so that the antiviral effect is stronger and longer than that of common drugs. At present, interferons are widely used clinically in China to treat chronic hepatitis B, chronic hepatitis C and the like.

Interferons can be divided into two major classes, type i and type ii IFNs, depending on their origin and structure: type I IFN includes 13 subtypes, such as IFN-alpha, IFN-beta, IFN-kappa, IFN-epsilon, IFN-omega, IFN-delta, etc., type II IFN is only one type of IFN-gamma (Bach EA, etc., 1997). In recent years, IFN-. lambda.1 (IL-28A), IFN-. lambda.2 (IL-28B), and IFN-. lambda.3 (IL-29) have been newly found to have interferon activity (Kotenko S V et al, 2003), but these bind to receptors different from type I interferon receptors, and are named as type III interferons.

Interferon kappa (IFN-. kappa.) is a recently discovered member of the type I interferon family, consisting of 207 amino acids, including a 27 amino acid signal peptide, and has 30% homology to other type I interferons, expressed by keratinocytes, monocytes, and Dendritic Cells (DCs), and exhibits tight tropism for keratinocytes and specific lymphoid cell populations (Decaro et al, 2010; LaFleur et al, 2001). IFN-. kappa.IFN- κ activates the expression of antiviral factors through the same signaling pathway as other type I interferons, i.e., by interacting with the IFN receptor (IFNR) 1/2. These genes regulate a wide range of cellular responses, including antiviral effects, antitumor effects, enhancement of NK cell activity, and activation of adaptive immune responses. Studies have shown that IFN- κ is selectively expressed in epithelial keratinocytes following viral infection, thereby inhibiting replication of encephalomyocarditis virus (ECMV) and Human Papilloma Virus (HPV) (decalo CA et al, 2010). In addition, IFN-kappa can inhibit the replication of various enveloped viruses including influenza virus, Zika virus and the like, and shows broad-spectrum antiviral effect.

One of the main components of the antiviral spray proposed by the public health clinical center of Shanghai city in 1 month 2020 is IFN-kappa, and from the data of laboratories, the novel coronavirus (SARS-CoV-2, 2019) is more sensitive to interferon than influenza virus and Zika virus, so the spray is likely to be more effective to coronavirus. Thus, IFN- κ has shown similar antiviral activity to other type I IFNs, but it is distinct from other type I IFNs in that it appears to signal in a discrete autocrine rather than paracrine cell-related manner (LaFleur et al, 2001). In current IFN treatment regimens, a number of side effects may limit the therapeutic potential of IFN, but due to the unique secretory properties of IFN- κ and the role of the regulatory immune molecules, current IFN treatment regimens may be improved and therefore have a better prospect. But due to their molecular nature, large-scale industrialization can be a great challenge, and optimization thereof is necessary.

Disclosure of Invention

In view of the problems of low yield, difficult denaturation and renaturation, uneven products and the like of IFN-kappa in the prior art due to the property of molecules, and difficulty in large-scale industrialization, the invention provides an IFN-kappa mutant and a preparation method thereof. The present invention may also solve one or more of the above problems.

An aspect of the present invention provides an interferon-kappa mutant, which is obtained by mutating a free cysteine residue to an amino acid other than cysteine and/or a methionine residue capable of forming an oxidative modification to an amino acid other than methionine, partially or entirely, based on a wild-type interferon-kappa.

Further, free cysteine residues are mutated to amino acids with smaller side chains; the methionine residue capable of forming oxidation modification is partially or totally mutated into one or more of amino acid with smaller side chain and/or hydrophobic amino acid.

Further, the amino acid having a smaller side chain is serine, alanine or glycine; the hydrophobic amino acid is valine, leucine or isoleucine.

In one embodiment, the interferon-kappa mutant further comprises one or more amino acids added to the N-terminus of the wild-type interferon-kappa.

Further, one or more amino acids added at the N-terminus are amino acids with smaller side chains.

Further, the amino acid having a smaller side chain is serine, alanine, or glycine.

Alternatively, the amino acid sequence of wild-type interferon- κ is as shown in SEQ ID No.1, or comprises the amino acid sequence as shown in SEQ ID No.1, or comprises an amino acid sequence at least 80%, 90%, 95%, 98%, 99% identical to SEQ ID No. 1.

Further, the free cysteine residue is cysteine residue 166 of the sequence shown in SEQ ID NO. 1; the methionine residues capable of forming oxidative modification are methionine residues at positions 52, 112, 115 and 120 of the sequence shown in SEQ ID NO. 1.

In one embodiment, the amino acid sequence of the interferon- κ mutant is as follows:

1) on the basis of the amino acid sequence shown in SEQ ID NO.1, the 166 th cysteine residue is replaced by serine, alanine or glycine; IFN-. kappa.s (C166S/G/A);

2) on the basis of the amino acid sequence shown in SEQ ID NO.1, the 166 th cysteine residue is replaced by serine, alanine or glycine, and serine, alanine or glycine is added at the N terminal; namely IFN-kappa (-1S/G/A, C166S/G/A);

3) on the basis of the amino acid sequence shown in SEQ ID No.1, the 166 th cysteine residue is replaced by serine, alanine or glycine, the N-terminal is added with serine, alanine or glycine, and the 112 th methionine residue and the 115 th methionine residue are replaced by serine, alanine, glycine or hydrophobic amino acid; IFN-kappa (-1S/G/A, M112, M115, C166S/G/A);

4) on the basis of the amino acid sequence shown in SEQ ID NO.1, the 166 th cysteine residue is replaced by serine, alanine or glycine, the N-terminal is added with serine, alanine or glycine, and the 115 th methionine residue is replaced by serine, alanine, glycine or hydrophobic amino acid; namely IFN-kappa (-1S/G/A, M115, C166S/G/A);

5) on the basis of the amino acid sequence shown in SEQ ID No.1, the 166 th cysteine residue is replaced by serine, alanine or glycine, the N-terminal is added with serine, alanine or glycine, and the 115 th methionine residue and the 120 th methionine residue are replaced by serine, alanine, glycine or hydrophobic amino acid; i.e., IFN-. kappa (-1S/G/A, C166S/G/A, M115, M120);

6) on the basis of the amino acid sequence shown in SEQ ID No.1, the 166 th cysteine residue is replaced by serine, alanine or glycine, the N-terminal is added with serine, alanine or glycine, and the 52 nd methionine residue, the 115 th methionine residue and the 120 th methionine residue are replaced by serine, alanine, glycine or hydrophobic amino acid; namely IFN-kappa (-1S/G/A, C166S/G/A, M52, M115, M120);

7) comprising an amino acid sequence as set forth in any one of 1) to 6); or

8) Comprising an amino acid sequence which is at least 80%, 90%, 95%, 98%, 99% identical to the amino acid sequence shown in any one of 1) to 6).

In a second aspect the invention provides an isolated polynucleotide, which in a particular embodiment encodes an interferon-kappa mutant as described above.

Alternatively, the sequence of the polynucleotide is as shown in SEQ ID No.4 or 6, or comprises the polynucleotide sequence as shown in SEQ ID No.4 or 6, or comprises a polynucleotide sequence that is at least 80%, 90%, 95%, 98%, 99% identical to the polynucleotide sequence of SEQ ID No.4 or 6.

In a third aspect, the present invention provides an expression vector, in a specific embodiment, comprising an isolated polynucleotide as described above.

In a fourth aspect, the invention provides a host cell, in a particular embodiment, comprising an isolated polynucleotide as described above or an expression vector as described above.

In a fifth aspect, the present invention provides a method for preparing an interferon-kappa mutant, which, in one embodiment, is as described above; the preparation method comprises the following steps: the polynucleotide sequence encoding the interferon-kappa mutant as described above is expressed in an expression system or expressed using an expression vector as described above or by a host cell as described above.

Optionally, the expression system is a prokaryotic expression system.

Alternatively, the polynucleotide sequence is ligated into an expression vector pET21b or pET41a and expressed in E.coli BL21(DE 3).

Further, the preparation method also comprises the following steps: the inclusion body of interferon-kappa mutant obtained by expression is subjected to in vitro renaturation and purification.

Optionally, in vitro renaturation is carried out by dissolving the inclusion body by using guanidine hydrochloride and/or DTT and renaturing in a renaturation solution; the purification is ion exchange chromatography purification and/or reverse phase chromatography purification.

In a fifth aspect, the invention provides the use of an interferon-kappa mutant for the manufacture of a medicament or formulation for the treatment of disease, the interferon-kappa mutant being as described above or being prepared by a method of manufacture as described above.

The interferon-kappa mutants in the embodiments of the present invention have the following advantages:

1) the free cysteine in the wild interferon-kappa is mutated into other amino acid residues, such as amino acid residues with smaller side chains, which is beneficial to the in vitro renaturation and purification of the expressed protein.

2) Partial or complete mutation of methionine residue capable of forming oxidation modification in wild-type interferon-kappa into other amino acid residues, such as amino acid residue with smaller side chain and/or hydrophobic amino acid, prevents or reduces the phenomenon of product heterogeneity caused by oxidation modification (oxidation) of methionine in subsequent in vitro renaturation and purification.

3) When E.coli is used for protein expression, the methionine residue generated from the initiation codon is not or hardly dropped, resulting in a phenomenon of heterogeneity of the product due to oxidative modification (oxidation) of methionine in subsequent in vitro renaturation and purification. Therefore, one or more amino acid residues are added after the initiation codon and before the first leucine residue (N-terminal) of the wild-type interferon-. kappa.to help the drop of methionine residue generated from the initiation codon, further avoiding the phenomenon of product heterogeneity.

4) The interferon in the embodiment of the present invention has the effect similar to that of IFN beta on the protection of WISH cells from Vesicular Stomatitis Virus (VSV).

Drawings

FIG. 1 is an electrophoretogram of a PCR product of IFN-. kappa.C 166S in example 1 of the present invention. Wherein M is a DNA molecular weight gradient; lanes 1 and 2 are the first and second round PCR products, respectively.

FIG. 2 is an electrophoretogram of the PCR product of IFN-. kappa. (-1S, M112A, M115A, C166S) in example 1 of the present invention.

Wherein M is Marker; lanes 1 and 2 are the first and second round PCR products, respectively.

FIG. 3 is an SDS-PAGE electrophoresis of IFN-. kappa. (wt) protein expressed in E.coli in example 2 of the present invention. Wherein, M is a protein standard; t is holomycoprotein; s is bacterial lysis supernatant; p is inclusion body.

FIG. 4 is an SDS-PAGE of the expression of IFN-. kappa.C 166S protein in E.coli in example 2 of the present invention. Wherein, M is a protein standard; t is holomycoprotein; s is bacterial lysis supernatant; p is inclusion body.

FIG. 5 is an SDS-PAGE of IFN-. kappa (-1S, M112A, M115A, C166S) protein expressed in E.coli in example 2 of the present invention. Wherein, M is a protein standard; t is holomycoprotein; s is bacterial lysis supernatant; p is inclusion body.

FIG. 6 is an SDS-PAGE of IFN-. kappa.wt and IFN-. kappa.C 166S inclusion bodies after renaturation in example 3 of the present invention using a 15% gradient gel. Where M is a protein standard (in kD), reducing means reducing agent in the loading buffer, and non-reducing means no reducing agent.

FIG. 7 is an SDS-PAGE electrophoresis of IFN-. kappa.C 166S inclusion body renaturation and purification in example 3 of the present invention, using 4-15% gradient gel. Where M is a protein standard (in kD), reducing means reducing agent in the loading buffer, and non-reducing means no reducing agent.

FIG. 8 is an SDS-PAGE of IFN-. kappa (-1S, M112A, M115A, C166S) inclusion body renaturation and purification in example 3 of the present invention, using 15% gel. Where M is a protein standard (in kD), reducing means reducing agent in the loading buffer, and non-reducing means no reducing agent.

FIG. 9 is a graph showing the results of identifying IFN-. kappa.s (C166S) by LC-MS.

FIG. 10 is a graph showing the results of identifying IFN-. kappa.1S, M112A, M115A, C166S by LC-MS.

FIG. 11 is a measurement of the protective effect of IFN-. kappa.C 166S on WISH cells; wherein, the positive control is IFN beta, and the negative control is a protein sample irrelevant to the type I interferon.

Detailed Description

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and are not intended to limit the scope of the present invention.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The reagents used are commercially available or publicly available reagents unless otherwise specified.

Hereinafter, unless otherwise stated, the amino acid sequence of the wild-type interferon-. kappa.is shown in SEQ ID NO.1, which is a protein _ ID in NCBI: the amino acid sequence of NP-064509 is formed by removing the signal peptide at positions 1-27.

Unless otherwise stated, the amino acid position in the amino acid sequence of the interferon-kappa mutant is calculated based on the amino acid sequence shown in SEQ ID No.1, i.e., the position of the first leucine of SEQ ID No.1 is 1 and the amino acid sequence after the first leucine is calculated; when other amino acids are added to the interferon-. kappa.mutant before the first leucine (N-terminal), the positions of the added other amino acids are calculated as-1 and-2 in this order (the position closest to the first leucine is-1).

"free cysteine residues" refers to cysteine residues that do not form disulfide bonds in interferon- κ.

"methionine residues capable of forming oxidative modifications" refers to methionine residues that may be exposed on the surface of a protein based on spatial conformation and surface amino acid analysis.

The relative sizes of the side chains at the amino acids are well known in the art and can be compared using any known metric, including steric effects, electron density, and the like. An example of an amino acid listed in order of increasing size is G, A, S, C, V, T, P, I, L, D, N, E, Q, M, K, H, F, Y, R, W.

Hydrophobic amino acids are well known in the art, such as V, L, I.

The interferon-kappa mutants according to one embodiment of the present invention are formed by mutating free cysteine residues to amino acids other than cysteine and/or partial or total methionine residues capable of forming oxidative modifications to amino acids other than methionine on the basis of wild-type interferon-kappa (which may be an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence comprising an amino acid sequence as shown in SEQ ID NO.1, or an amino acid sequence having at least 80%, 90%, 95%, 98%, 99% identity to SEQ ID NO. 1). In some embodiments, the free cysteine residue is mutated to an amino acid with a smaller side chain; the methionine residue capable of forming oxidation modification is partially or completely mutated into amino acid with smaller side chain and/or hydrophobic amino acid. Amino acids with smaller side chains include, but are not limited to, glycine, alanine, serine, valine, threonine. Hydrophobic amino acids include, but are not limited to, valine, leucine, isoleucine. The free cysteine residue is mutated into an amino acid residue with a smaller side chain, which is beneficial to the in vitro renaturation and purification of the subsequent expressed protein. Partial or complete mutation of methionine residues capable of forming oxidative modification into amino acid residues with smaller side chains and/or hydrophobic amino acids can prevent or reduce the phenomenon of non-uniformity of products caused by oxidative modification (oxidation) of methionine in subsequent in vitro renaturation and purification.

In one embodiment, the free cysteine residue is cysteine residue 166 of the sequence shown in SEQ ID NO. 1; the methionine residues capable of forming oxidative modification are methionine residues at positions 52, 112, 115 and 120 of the sequence shown in SEQ ID NO. 1.

In another embodiment, the interferon-kappa mutant may be expressed by an expression system. The expression system may be a prokaryotic expression system, a eukaryotic expression system or a cell-free expression system. In order to avoid or reduce the phenomenon of non-uniformity of the product due to the formation of oxidative modification (oxidation) of the methionine residue after translation of the start codon when expressed using prokaryotic expression systems, such as E.coli expression systems, after translation of the methionine residue formed by the start codon, one or more amino acid residues, such as one, two or three amino acid residues, are added to facilitate methionine cleavage catalyzed by Methionine Aminopeptidase (MAP), in order to avoid or reduce the phenomenon that the methionine residue formed after translation of the start codon does not fall off and the product is not uniform during subsequent in vitro renaturation and purification. The one or more amino acid residues added are not methionine residues, not cysteine residues, optionally amino acid residues with smaller side chains, including but not limited to glycine, alanine, serine, valine, threonine.

In another embodiment, one or two or three of the three improvements can be made to wild-type interferon- κ (which may be the amino acid sequence shown in SEQ ID No.1, or comprise an amino acid sequence at least 80%, 90%, 95%, 98%, 99% identical to SEQ ID No. 1): 1) mutating a free cysteine residue to an amino acid other than cysteine, 2) mutating a methionine residue capable of forming an oxidative modification partially or completely to an amino acid other than methionine, 3) adding one or more amino acid residues after translation of the methionine residue formed at the start codon.

In the following examples, the amino acid sequence of wild-type interferon-. kappa.is shown in SEQ ID NO. 1.

Example 1 design of recombinant IFN-. kappa.and construction of expression plasmid

IFN-kappa wt is wild type interferon-kappa, the amino acid sequence of which is shown as SEQ ID NO.1, and the nucleotide sequence of which is shown as SEQ ID NO. 2.

To facilitate in vitro renaturation and purification of IFN- κ and/or to make the product unique, the following recombinant IFN- κ was designed:

IFN-. kappa (-1S/G/A, C166S/G/A), such as IFN-. kappa (-1S, C166S);

IFN- κ (C166S/G/A), e.g., IFN- κ (C166S);

IFN- κ (-1S, M112, M115, C166S), e.g. IFN- κ (-1S, M112A, M115A, C166S), IFN- κ (-1S, M112V, M115A, C166S);

IFN- κ (-1S, M115, C166S), e.g. IFN- κ (-1S, M115A, C166S), IFN- κ (-1S, M115V, C166S);

IFN- κ (-1S, C166S, M115, M120), e.g. IFN- κ (-1S, C166S, M115A, M120A), IFN- κ (-1S, C166S, M115A, M120L);

IFN- κ (-1S, C166S, M52, M115, M120), e.g. IFN- κ (-1S, C166S, M52S, M115G, M120A), IFN- κ (-1S, C166S, M52S, M115V, M120L).

Wherein represents an amino acid with a smaller side chain or a hydrophobic amino acid.

IFN-kappa (C166S) is based on wild interferon-kappa, the 166 th cysteine is mutated into serine, the amino acid sequence is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4.

IFN-kappa (-1S, M112A, M115A, C166S) is based on wild-type interferon-kappa, and a serine is added before leucine at position 1 (-position 1), methionine at positions 112 and 115 is mutated into alanine, cysteine at position 166 is mutated into serine, the amino acid sequence is shown as SEQ ID NO.5, and the nucleotide sequence is shown as SEQ ID NO. 6.

1.1 Gene and primer Synthesis

The nucleotide sequence of IFN-. kappa.wt (shown as SEQ ID NO. 2) was synthesized by Zhongmeitai and Biotechnology (Beijing) Inc.

The PCR primers of SEQ ID NO.4 and SEQ ID NO.6 were synthesized by Shanghai Biotechnology engineering Co., Ltd, and are specifically shown in Table 1:

TABLE 1 primer sequences

1.2 construction of expression plasmids

Carrying out 2-round PCR amplification (respectively taking SEQ4-F1, SEQ4-R1, SEQ4-F1 and SEQ4-R2 as primers) by taking the gene SEQ ID NO.2 as a template, carrying out DNA gel recovery on a PCR product obtained by amplification (shown in figure 1), and then recombining into a pET21b plasmid to construct a pET21b-IFNK C166S expression plasmid; and using the obtained pET21b-IFNK (C166S) expression plasmid as a template, carrying out 2-round PCR amplification (respectively using SEQ6-F1, SEQ6-R1, SEQ6-F2 and SEQ6-R2 as primers), carrying out DNA gel recovery on the PCR product obtained by amplification (as shown in figure 2), and constructing pET41a-IFNK (-1S, M112A, M115A and C166S) expression plasmid. The above procedures were performed according to molecular cloning.

Meanwhile, pET21b-IFNK (wt) expression plasmid is constructed.

Example 2 expression of IFN-. kappa.wt, recombinant IFN-. kappa.C 166S and IFN-. kappa.1S, M112A, M115A, C166S in E.coli

mu.L of the expression plasmid prepared in example 1 was used to transform expression strain BL21(DE3), and 1 single colony was selected for bacterial conservation.

According to the following steps: 2000 ratio, the next day according to 1: 50 ratio transfer to Amp resistance containing 500mL LB medium triangle bottle, 37 degrees, 220rpm, cultured to OD₆₀₀When the concentration is 2, 0.5mM IPTG is added, and the strain is harvested after induction for 3h at 37 degrees. The harvested thalli is suspended by buffer solution, high-pressure crushing is carried out, 800bar is carried out for 3 times, and then SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) reductive electrophoresis detection is carried out on the expressed protein to check whether the protein with the correct size is generated. The above procedures were performed according to molecular cloning.

As shown in FIG. 3, FIG. 4 and FIG. 5, IFN-. kappa.wt, IFN-. kappa.C 166S and IFN-. kappa.C (-1S, M112A, M115A and C166S) all showed that specific proteins were expressed between 20kD and 25kD, and the target protein was mainly expressed in the form of inclusion bodies.

Example 3 in vitro renaturation and purification of recombinant IFN-. kappa.wt, IFN-. kappa.C 166S and IFN-. kappa.1S, M112A, M115A, C166S

Inclusion bodies used in vitro renaturation and purification were obtained using the culture, induction and disruption method as in example 2.

3.1 Inclusion body solubilization and renaturation

The inclusion bodies containing the target protein obtained in example 2 were each solubilized with 6M guanidine hydrochloride and 20mM DTT, and then the solubilized inclusion bodies were each diluted 1:100 into a renaturation solution (20mM Tris-HCl, 1mM cysteine (cysteine), 1mM cystine (cystine), 0.1% SDS, pH9.0) overnight at room temperature as a renaturation sample, i.e., a renaturation sample of IFN-. kappa.wt, IFN-. kappa.C 166S or IFN-. kappa.1S, M112A, M115A, C166S).

As shown in FIG. 6, IFN-. kappa.C 166S showed specific target bands on both reducing and non-reducing gels, and the folded target protein migrated faster under non-reducing conditions than under reducing conditions; whereas IFN-. kappa.wt only shows the band of interest under reducing conditions, but not in principle, and may form a precipitate or be a polymer.

3.2 purification by ion exchange chromatography

The renatured sample is centrifuged for 30 minutes at 10000g, the supernatant is loaded on a 20ml cation exchange column (purchased from GE healthcare), then NaCl gradient elution is carried out, and a collection tube where the target protein is located is selected according to SDS-PAGE and is combined for storage, so that the target protein can be captured and some foreign proteins can be removed.

3.3 purification by reverse phase chromatography

The pooled proteins after ion exchange chromatography were passed through reverse phase chromatography column C8 (from YMC) using an acetonitrile gradient to remove folding errors and some modified proteins.

3.4 ion exchange Fine purification

The combined proteins after reverse phase chromatography column C8 were suitably diluted for cation exchange fine purification, eluting with a NaCL gradient to remove some contaminating proteins.

3.5 desalting and liquid changing of desalting column

IFN-. kappa.protein was desalted and exchanged using desalting column G25 (from GE healthcare) and final buffer PBS, pH 7.4.

The SDS-PAGE results of the final samples are shown in FIGS. 7 and 8 as a single band, and the folded target protein migrates faster under non-reducing conditions than under reducing conditions.

3.6 LC-MS identification

IFN-. kappa.C166S had a theoretical molecular weight of 22306 (with an initial Met), whereas IFN-. kappa.C166S in this example had a correct molecular weight (FIG. 9) and was successfully renatured but also had a continuous molecular weight of +16, suggesting an oxidative modification (oxidation) resulting in product heterogeneity, as identified by LC-MS (positive ion mode, 70V; Capillary voltage: 1.5 kV; mass range:400-7000 m/z); the theoretical molecular weight of the mutated IFN-kappa (-1S, M112A, M115A and C166S) is 22142, the actually measured molecular weight is correct (as shown in figure 10), no oxidation modification is carried out, and the product is more uniform.

EXAMPLE 4 Activity assay of IFN-. kappa.C 166S after renaturation and purification

The invention utilizes the 2015 edition of Chinese pharmacopoeia, three ministry of general rules 3523 interferon biological activity determination method to detect the activity of interferon, this method is according to interferon can protect human amniotic cells (WISH) from Vesicular Stomatitis Virus (VSV) destructive effect, use MTT method to stain surviving WISH cells, measure its absorbance in the enzyme labeling instrument, can get interferon to WISH cell protective effect curve, thus determine interferon biological activity. Wherein the highest concentration of IFN-kappa (C166S) used was 10000ng/mL, and the dilution was performed by 5-fold gradient, and the total dilution was 10.

As shown in FIG. 11, the IFN-. kappa.C 166S sample served to significantly protect WISH cells from Vesicular Stomatitis Virus (VSV), and the IFN-. kappa.C 166S sample was effective at a dose of 5ng/ml, and at the respective doses of maximal function, the protective effect on WISH cells was comparable to that of the positive control IFN-. beta.s.

SEQUENCE LISTING

<110> Beijing Zhidao Biotechnology Co., Ltd

<120> interferon-kappa mutant and preparation method thereof

<150> CN202010425805.6

<151> 2020-05-19

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 180

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Leu Asp Cys Asn Leu Leu Asn Val His Leu Arg Arg Val Thr Trp Gln

1 5 10 15

Asn Leu Arg His Leu Ser Ser Met Ser Asn Ser Phe Pro Val Glu Cys

20 25 30

Leu Arg Glu Asn Ile Ala Phe Glu Leu Pro Gln Glu Phe Leu Gln Tyr

35 40 45

Thr Gln Pro Met Lys Arg Asp Ile Lys Lys Ala Phe Tyr Glu Met Ser

50 55 60

Leu Gln Ala Phe Asn Ile Phe Ser Gln His Thr Phe Lys Tyr Trp Lys

65 70 75 80

Glu Arg His Leu Lys Gln Ile Gln Ile Gly Leu Asp Gln Gln Ala Glu

85 90 95

Tyr Leu Asn Gln Cys Leu Glu Glu Asp Lys Asn Glu Asn Glu Asp Met

100 105 110

Lys Glu Met Lys Glu Asn Glu Met Lys Pro Ser Glu Ala Arg Val Pro

115 120 125

Gln Leu Ser Ser Leu Glu Leu Arg Arg Tyr Phe His Arg Ile Asp Asn

130 135 140

Phe Leu Lys Glu Lys Lys Tyr Ser Asp Cys Ala Trp Glu Ile Val Arg

145 150 155 160

Val Glu Ile Arg Arg Cys Leu Tyr Tyr Phe Tyr Lys Phe Thr Ala Leu

165 170 175

Phe Arg Arg Lys

180

<210> 2

<211> 540

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ctggactgca acctgctgaa tgtccacctg cgtcgcgtta cctggcagaa cctgcgtcat 60

ctgagttcta tgagcaatag ttttccagtt gagtgcctgc gtgagaacat tgccttcgaa 120

ctgcctcagg agtttctgca atacacgcag ccgatgaagc gcgacatcaa aaaggctttc 180

tacgaaatga gtctgcaagc gtttaacatc ttctctcaac atactttcaa gtactggaaa 240

gaacgtcacc tgaaacaaat ccaaattggt ctggaccagc aggctgagta cctgaatcag 300

tgtctggaag aagacaaaaa cgagaacgaa gacatgaagg agatgaaaga aaatgagatg 360

aagccaagcg aagctcgcgt tccacagctg agtagcctgg agctgcgtcg ctactttcat 420

cgtatcgaca atttcctgaa ggaaaagaag tactctgatt gtgcttggga aattgttcgc 480

gttgaaattc gtcgttgcct gtactacttt tacaaattca ccgctctgtt tcgtcgcaaa 540

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Asn Leu Arg His Leu Ser Ser Met Ser Asn Ser Phe Pro Val Glu Cys

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Leu Arg Glu Asn Ile Ala Phe Glu Leu Pro Gln Glu Phe Leu Gln Tyr

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Thr Gln Pro Met Lys Arg Asp Ile Lys Lys Ala Phe Tyr Glu Met Ser

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Leu Gln Ala Phe Asn Ile Phe Ser Gln His Thr Phe Lys Tyr Trp Lys

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Glu Arg His Leu Lys Gln Ile Gln Ile Gly Leu Asp Gln Gln Ala Glu

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Tyr Leu Asn Gln Cys Leu Glu Glu Asp Lys Asn Glu Asn Glu Asp Met

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Lys Glu Met Lys Glu Asn Glu Met Lys Pro Ser Glu Ala Arg Val Pro

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Phe Leu Lys Glu Lys Lys Tyr Ser Asp Cys Ala Trp Glu Ile Val Arg

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Phe Arg Arg Lys

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cgcattgaca actttctgaa ggagaagaag tatagtgact gtgcctggga aatcgttcgt 480

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Ser Leu Asp Cys Asn Leu Leu Asn Val His Leu Arg Arg Val Thr Trp

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Gln Asn Leu Arg His Leu Ser Ser Met Ser Asn Ser Phe Pro Val Glu

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Cys Leu Arg Glu Asn Ile Ala Phe Glu Leu Pro Gln Glu Phe Leu Gln

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Tyr Thr Gln Pro Met Lys Arg Asp Ile Lys Lys Ala Phe Tyr Glu Met

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Ser Leu Gln Ala Phe Asn Ile Phe Ser Gln His Thr Phe Lys Tyr Trp

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aaagaacgtc acctgaaaca gatccaaatc ggcctggatc aacaggccga gtacctgaat 300

caatgtctgg aagaggacaa gaatgaaaat gaggacgcga aagaagcaaa agagaacgaa 360

atgaagccat ctgaagcccg tgtaccgcaa ctgagtagtc tggagctgcg tcgctacttt 420

catcgtatcg acaacttcct gaaagagaag aaatacagtg actgcgcgtg ggagatcgta 480

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aag 543

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<212> DNA

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gaggatgcga aagaagcgaa agagaacgag atgaaaccgt c 41

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gtggtggtgg tggtgctcga gttatttacg acggaacaga gcagt 45