WO2021233094A1 - 干扰素-κ突变体及其制备方法 - Google Patents

干扰素-κ突变体及其制备方法 Download PDF

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WO2021233094A1
WO2021233094A1 PCT/CN2021/090472 CN2021090472W WO2021233094A1 WO 2021233094 A1 WO2021233094 A1 WO 2021233094A1 CN 2021090472 W CN2021090472 W CN 2021090472W WO 2021233094 A1 WO2021233094 A1 WO 2021233094A1
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interferon
amino acid
mutant
serine
alanine
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French (fr)
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赵耀
张建军
魏婷婷
孟万利
张秋磊
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北京志道生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
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    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of molecular biology, and specifically relates to an interferon- ⁇ mutant and a preparation method thereof.
  • Interferon is an anti-influenza virus factor (Isaacs et al., 1957) discovered by scientists Isaacs and Lindenmann in 1957 that chicken embryos infected with influenza virus (Isaacs et al., 1957), a protein with a wide range of biological activities It is an important cytokine that can be synthesized and secreted by a variety of cells. It has multiple functions such as anti-virus, anti-tumor and immune regulation (Takaoka A et al., 2003). It is an important part of the body's natural immune defense system.
  • Interferon is an inducing protein, which is generally not produced spontaneously by normal cells, and belongs to the immune system that exerts the fastest virus defense function.
  • the antiviral effect of interferon is often earlier than the specific immune response of the body, can effectively limit virus replication, and has the activity of inhibiting tumor cell proliferation and regulating immune function (LaFleur et al., 2001; Nardelli et al., 2002), and is worldwide The approved drugs for the treatment of a variety of viral diseases, tumors and immune disorders.
  • the current antiviral drugs only have a pure antiviral effect, while IFN has the dual effects of antiviral and regulating immune function at the same time, so the antiviral effect is stronger and lasting than general drugs.
  • interferon is widely used clinically in my country to treat chronic hepatitis B and chronic hepatitis C.
  • interferons can be divided into two categories: Type I IFN and Type II IFN: Type I IFN includes IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , etc. Of the 13 subtypes, type II IFN has only one IFN- ⁇ (Bach EA et al., 1997). In recent years, there have been newly discovered IFN- ⁇ 1 (IL-28A), IFN- ⁇ 2 (IL-28B), IFN- ⁇ 3 (IL-29) (Kotenko S V, etc., 2003) with interferon activity, but they The bound receptor is different from the type I interferon receptor, so it is named type III interferon.
  • Interferon- ⁇ (interferon kappa, IFN- ⁇ ) is a recently discovered member of the type I interferon family. It consists of 207 amino acids, including a signal peptide of 27 amino acids. IFN- ⁇ and other type I interferons have 30% The homology of keratinocytes, monocytes and dendritic cells (DC) is expressed by keratinocytes, monocytes and dendritic cells (DC), and shows a compact tropism to keratinocytes and specific lymphoid cell populations (DeCarlo et al., 2010; LaFleur et al. People, 2001).
  • IFN- ⁇ can activate the expression of antiviral factors through the same signaling pathway as other type I interferons, that is, interacting with IFN receptor (IFNR) 1/2. These genes regulate a wide range of cellular responses, including antiviral effects, anti-tumor effects, enhancement of NK cell activity, and activation of adaptive immune responses. Studies have shown that after viral infection, IFN- ⁇ is selectively expressed in epithelial keratinocytes, thereby inhibiting the replication of encephalomyocarditis virus (ECMV) and human papilloma virus (HPV) (DeCarlo CA et al., 2010). In addition, IFN- ⁇ can also inhibit the replication of a variety of enveloped viruses, including influenza virus, Zika virus, etc., showing a broad-spectrum antiviral effect.
  • ECMV encephalomyocarditis virus
  • HPV human papilloma virus
  • IFN- ⁇ One of the main components of the antiviral spray proposed by the Shanghai Public Health Clinical Center in January 2020 is IFN- ⁇ .
  • the new coronavirus SARS-CoV-2, 2019
  • Zika virus are more sensitive to interferon, so sprays may be more effective against coronavirus. Therefore, IFN- ⁇ has shown similar antiviral activity to other type I IFNs, but it is very different from other type I IFNs because it seems to signal in discrete autocrine rather than paracrine cell-related ways (LaFleur et al. ,2001).
  • the present invention provides an IFN- ⁇ mutant and ⁇ The method of preparation.
  • the present invention can also solve only one or several of the above-mentioned problems.
  • the interferon- ⁇ mutant combines free cysteine residues on the basis of wild-type interferon- ⁇ .
  • the base is mutated to an amino acid other than cysteine, and/or part or all of the methionine residues capable of forming an oxidative modification are mutated to an amino acid other than methionine.
  • mutate free cysteine residues to amino acids with smaller side chains mutate part or all of methionine residues capable of forming oxidative modifications to amino acids with smaller side chains and/or hydrophobic amino acids One or more of.
  • amino acid with a smaller side chain is serine, alanine or glycine; the hydrophobic amino acid is valine, leucine or isoleucine.
  • the interferon- ⁇ mutant further adds one or more amino acids to the N-terminal on the basis of wild-type interferon- ⁇ .
  • amino acids added at the N-terminus are amino acids with smaller side chains.
  • amino acid with a smaller side chain is serine, alanine or glycine.
  • amino acid sequence of wild-type interferon- ⁇ is shown in SEQ ID NO. 1, or contains the amino acid sequence shown in SEQ ID NO. 1, or contains at least 80%, 90% of the amino acid sequence shown in SEQ ID NO.1. , 95%, 98%, 99% identical amino acid sequences.
  • the free cysteine residue is the 166th cysteine residue of the sequence shown in SEQ ID NO. 1; the methionine residue capable of forming an oxidative modification is the cysteine residue of SEQ ID NO. 1.
  • amino acid sequence of the interferon- ⁇ mutant is as follows:
  • the second aspect of the present invention provides an isolated polynucleotide, which in a specific embodiment encodes the interferon- ⁇ mutant as described above.
  • 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 as shown in SEQ ID NO. 4 or 6.
  • the nucleotide sequence is at least 80%, 90%, 95%, 98%, 99% identical to a polynucleotide sequence.
  • the third aspect of the present invention provides an expression vector, in a specific embodiment, comprising an isolated polynucleotide as described above.
  • the fourth aspect of the present invention provides a host cell, in a specific embodiment, comprising an isolated polynucleotide as described above or an expression vector as described above.
  • the fifth aspect of the present invention provides a method for preparing an interferon- ⁇ mutant.
  • the interferon- ⁇ mutant is described above; the preparation method is: expressing the code in an expression system as described above
  • the polynucleotide sequence of the interferon- ⁇ mutant can be expressed either by using an expression vector as described above, or expressed by a host cell as described above.
  • the expression system is a prokaryotic expression system.
  • polynucleotide sequence is linked to the expression vector pET21b or pET41a and expressed in E. coli BL21 (DE3).
  • the preparation method further includes: renaturing and purifying the inclusion bodies obtained by expressing the interferon- ⁇ mutant in vitro.
  • the in vitro renaturation is the use of guanidine hydrochloride and/or DTT to dissolve the inclusion bodies and the renaturation in the renaturation solution;
  • the purification is ion exchange chromatography purification and/or reverse chromatography purification.
  • the fifth aspect of the present invention provides the use of the interferon- ⁇ mutant in the preparation of drugs or preparations for the treatment of diseases.
  • the interferon- ⁇ mutant is as described above, or is prepared by the above-mentioned preparation method.
  • the interferon- ⁇ mutant in the specific embodiment of the present invention has the following advantages:
  • the interferon in the specific embodiment of the present invention has a similar effect to IFN ⁇ in protecting WISH cells from damage by vesicular stomatitis virus (VSV).
  • VSV vesicular stomatitis virus
  • Figure 1 is an electrophoresis diagram of the PCR product of IFN- ⁇ (C166S) in Example 1 of the present invention.
  • M is the DNA molecular weight gradient
  • Lane 1 and Lane 2 are the products of the first round of PCR and the second round of PCR, respectively.
  • Figure 2 is an electrophoresis diagram of PCR products of IFN- ⁇ (-1S, M112A, M115A, C166S) in Example 1 of the present invention.
  • M is Marker
  • Lane 1 and Lane 2 are the products of the first round of PCR and the second round of PCR, respectively.
  • Fig. 3 is an SDS-PAGE electrophoresis diagram of the expression of IFN- ⁇ (wt) protein in E. coli in Example 2 of the present invention.
  • M is the protein standard
  • T is the whole bacterial protein
  • S is the bacterial lysate supernatant
  • P is the inclusion body.
  • FIG. 4 is an SDS-PAGE electrophoresis diagram of the expression of IFN- ⁇ (C166S) protein in E. coli in Example 2 of the present invention.
  • M is the protein standard
  • T is the whole bacterial protein
  • S is the bacterial lysate supernatant
  • P is the inclusion body.
  • Figure 5 is an SDS-PAGE electrophoresis diagram of IFN- ⁇ (-1S, M112A, M115A, C166S) protein expressed in E. coli in Example 2 of the present invention.
  • M is the protein standard
  • T is the whole bacterial protein
  • S is the bacterial lysate supernatant
  • P is the inclusion body.
  • Fig. 6 is an SDS-PAGE electrophoresis diagram of IFN- ⁇ (wt) and IFN- ⁇ (C166S) inclusion bodies after renaturation in Example 3 of the present invention, using a 15% gradient gel.
  • M is the protein standard (unit kD)
  • reduction means that there is a reducing agent in the loading buffer
  • non-reduction means that there is no reducing agent.
  • Figure 7 is an SDS-PAGE electrophoresis diagram of IFN- ⁇ (C166S) inclusion bodies after renaturation and purification in Example 3 of the present invention, using a 4-15% gradient gel.
  • M is the protein standard (unit kD)
  • reduction means that there is a reducing agent in the loading buffer
  • non-reduction means that there is no reducing agent.
  • Figure 8 is an SDS-PAGE electrophoresis diagram of IFN- ⁇ (-1S, M112A, M115A, C166S) inclusion bodies after renaturation and purification in Example 3 of the present invention, using 15% gel.
  • M is the protein standard (unit kD)
  • reduction means that there is a reducing agent in the loading buffer
  • non-reduction means that there is no reducing agent.
  • Figure 9 is a diagram showing the results of the identification of IFN- ⁇ (C166S) using LC-MS.
  • Figure 10 is a diagram of the results of the identification of IFN- ⁇ (-1S, M112A, M115A, C166S) using LC-MS.
  • Figure 11 is the determination of the protective effect of IFN- ⁇ (C166S) on WISH cells; wherein the positive control is IFN ⁇ , and the negative control is a protein sample not related to type I interferon.
  • amino acid sequence of wild-type interferon- ⁇ is shown in SEQ ID NO. 1, which is formed by removing the signal peptide at positions 1 to 27 from the amino acid sequence of protein_id: NP_064509 in NCBI.
  • the amino acid position in the amino acid sequence of the interferon- ⁇ mutant is calculated based on the amino acid sequence shown in SEQ ID NO. 1, that is, the position of the first leucine in SEQ ID NO. 1 is 1. , The position of the amino acid sequence after the first leucine is calculated; if the interferon- ⁇ mutant adds other amino acids before the first leucine (N-terminal), the positions of the added other amino acids will be -1, -2 is calculated in turn (the position closest to the first leucine is -1).
  • Free cysteine residues refer to cysteine residues that do not form disulfide bonds in interferon- ⁇ .
  • Methionine residues capable of forming oxidative modification refer to methionine residues that may be exposed on the surface of the protein based on the spatial conformation and surface amino acid analysis.
  • the relative sizes of amino acid side chains are well known in the art, and can be compared using any known metrics, including steric effects, electron density, and the like.
  • An example of amino acids 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- ⁇ mutant is obtained by adding wild-type interferon- ⁇ (which can be the amino acid sequence shown in SEQ ID NO.1, or contains the amino acid sequence shown in SEQ ID NO.1). On the basis of the amino acid sequence of SEQ ID NO.1, or an amino acid sequence that is at least 80%, 90%, 95%, 98%, 99% identical to SEQ ID NO. 1, by mutating free cysteine residues to half Amino acids other than cystine, and/or methionine residues capable of forming an oxidative modification are partially or completely mutated into amino acids other than methionine.
  • free cysteine residues are mutated into amino acids with smaller side chains; part or all of methionine residues capable of forming oxidative modifications are mutated into amino acids with smaller side chains and/or Hydrophobic amino acid.
  • Amino acids with smaller side chains include, but are not limited to, glycine, alanine, serine, valine, and threonine.
  • Hydrophobic amino acids include, but are not limited to, valine, leucine, and isoleucine. Mutation of free cysteine residues into amino acid residues with smaller side chains is helpful for the subsequent in vitro renaturation and purification of the expressed protein.
  • Mutation of part or all 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 subsequent in vitro renaturation and purification of methionine The formation of non-uniform products caused by oxidation.
  • the free cysteine residue is the 166th cysteine residue of the sequence shown in SEQ ID NO. 1; the methionine residue capable of forming an oxidative modification is SEQ ID The 52nd, 112th, 115th and 120th methionine residues of the sequence shown in NO.1.
  • the interferon- ⁇ mutant can be expressed by an expression system.
  • the expression system can be a prokaryotic expression system, a eukaryotic expression system or a cell-free expression system.
  • a prokaryotic expression system such as an E. coli expression system for expression
  • the methionine residue will be refolded and purified in vitro. Amino acid residues form the phenomenon of inhomogeneous products caused by oxidation.
  • one or more amino acid residues are added, such as adding one, two or Three amino acid residues to help methionylaminopeptidase (MAP) catalyze the excision of methionine.
  • the added one or more amino acid residues are not methionine residues, not cysteine residues, and optionally amino acid residues with smaller side chains, including but not limited to, glycine, alanine, and serine , Valine, threonine.
  • wild-type interferon- ⁇ (which can be the amino acid sequence shown in SEQ ID NO. 1, or contains the amino acid sequence shown in SEQ ID NO. 1, or contains the amino acid sequence shown in SEQ ID NO. NO.1 Amino acid sequences with at least 80%, 90%, 95%, 98%, 99% identity) one or two or three of the three improvements: 1) Free cysteine residues Mutated to amino acids other than cysteine, 2) mutated part or all of methionine residues capable of forming oxidative modification to amino acids other than methionine, 3) translated at the start codon After the formed methionine residue, one or more amino acid residues are added.
  • amino acid sequence of wild-type interferon- ⁇ is shown in SEQ ID NO.1.
  • IFN- ⁇ wt is wild-type interferon- ⁇ , and its amino acid sequence is shown in SEQ ID NO. 1, and its nucleotide sequence is shown in SEQ ID NO. 2.
  • IFN- ⁇ In order to facilitate the in vitro renaturation and purification of IFN- ⁇ and/or make the product single, the following recombinant IFN- ⁇ is designed:
  • IFN- ⁇ (-1S/G/A, C166S/G/A), such as IFN- ⁇ (-1S, C166S);
  • IFN- ⁇ C166S/G/A
  • IFN- ⁇ C166S
  • IFN- ⁇ (-1S, M112*, M115*, C166S), such as IFN- ⁇ (-1S, M112A, M115A, C166S), IFN- ⁇ (-1S, M112V, M115A, C166S);
  • IFN- ⁇ (-1S, M115*, C166S), such as IFN- ⁇ (-1S, M115A, C166S), IFN- ⁇ (-1S, M115V, C166S);
  • IFN- ⁇ (-1S, C166S, M115*, M120*), such as IFN- ⁇ (-1S, C166S, M115A, M120A), IFN- ⁇ (-1S, C166S, M115A, M120L);
  • IFN- ⁇ (C166S) is based on wild-type interferon- ⁇ , the cysteine at position 166 is mutated to serine. Its amino acid sequence is shown in SEQ ID NO. 3, and its nucleotide sequence is shown in SEQ ID NO.4 is shown.
  • IFN- ⁇ (-1S, M112A, M115A, C166S) is based on wild-type interferon- ⁇ , adding a serine before the first leucine (position -1), combining the 112th and 115th positions
  • the methionine was mutated to alanine, and the cysteine at position 166 was mutated to serine.
  • Its amino acid sequence is shown in SEQ ID NO. 5, and its nucleotide sequence is shown in SEQ ID NO. 6. Show.
  • the nucleotide sequence of IFN- ⁇ wt (shown in SEQ ID NO. 2) was synthesized by Zhongmeitaihe Biotechnology (Beijing) Co., Ltd.
  • the PCR primers of SEQ ID NO. 4 and SEQ ID NO. 6 were synthesized by Shanghai Shenggong Biological Engineering Co., Ltd., as shown in Table 1:
  • SEQ ID NO.2 gene As a template, two rounds of PCR amplification (using SEQ4-F1 and SEQ4-R1, SEQ4-F1 and SEQ4-R2 as primers respectively) were carried out, and the PCR products obtained by amplification were recovered by DNA gel ( As shown in Figure 1), the pET21b plasmid was then recombined to construct the pET21b-IFNK C166S expression plasmid; and the obtained pET21b-IFNK (C166S) expression plasmid was used as a template, and two rounds of PCR amplification were carried out (using SEQ6-F1 and SEQ6-F1, respectively).
  • SEQ6-R1, SEQ6-F2 and SEQ6-R2 are primers), and the amplified PCR product is recovered by DNA gel (as shown in Figure 2) to construct pET41a-IFNK (-1S, M112A, M115A, C166S) expression Plasmid.
  • pET41a-IFNK -1S, M112A, M115A, C166S
  • Example 2 Expression of IFN- ⁇ wt, recombinant IFN- ⁇ (C166S) and IFN- ⁇ (-1S, M112A, M115A, C166S) in E. coli
  • IFN- ⁇ wt, IFN- ⁇ (C166S) and IFN- ⁇ (-1S, M112A, M115A, C166S) all obtained specific proteins between 20kD and 25kD. Expression, and the target protein is mainly expressed in the form of inclusion bodies.
  • the inclusion bodies used in the in vitro renaturation and purification were obtained by the method of cultivation, induction and disruption as in Example 2.
  • Example 2 Dissolve the inclusion bodies containing the target protein obtained in Example 2 with 6M guanidine hydrochloride and 20mM DTT, and then dilute the dissolved inclusion bodies into the refolding solution at a ratio of 1:100 (20mM Tris-HCl, 1mM half Cystine (cysteine), 1mM cystine (cystine), 0.1% SDS, pH 9.0), overnight at room temperature, as a refolding sample, namely IFN- ⁇ wt, IFN- ⁇ (C166S) or IFN- ⁇ (-1S) , M112A, M115A, C166S) refolding samples.
  • a refolding sample namely IFN- ⁇ wt, IFN- ⁇ (C166S) or IFN- ⁇ (-1S) , M112A, M115A, C166S) refolding samples.
  • IFN- ⁇ (C166S) can see specific target bands on both reducing and non-reducing gels, and the folded target protein migrates faster under non-reducing conditions than under reducing conditions; IFN- ⁇ wt only sees the target band under reducing conditions, not in principle, it may be that a precipitate or a polymer is formed.
  • the refolded sample was centrifuged at 10000g for 30 minutes, and the supernatant was loaded on a 20ml cation exchange column (purchased from GE healthcare), and then subjected to NaCl gradient elution. According to SDS-PAGE, select the collection tube where the target protein is located and save it. This step can capture the target protein and remove some impurities.
  • the combined protein was passed through reversed-phase chromatography column C8 (purchased from YMC) and eluted with an acetonitrile gradient to remove folding errors and some modified proteins.
  • the combined protein was appropriately diluted and then subjected to cation exchange fine purification, and NaCL gradient elution was used to remove some impurity proteins.
  • Desalting column G25 (purchased from GE healthcare) was used to desalt the IFN- ⁇ protein and the final buffer was PBS, pH 7.4.
  • the theoretical molecular weight of IFN- ⁇ (C166S) is 22306 (with starting Met), and it has been identified by LC-MS (positive ion mode, cone voltage: 70V; Capillary voltage: 1.5kV; mass range: 400-7000m/z)
  • the molecular weight of IFN- ⁇ (C166S) is correct (as shown in Figure 9), and the renaturation is successful, but there are also continuous molecular weights of +16, suggesting oxidation, which causes unevenness in the product; and after mutation
  • the theoretical molecular weight of the latter IFN- ⁇ (-1S, M112A, M115A, C166S) is 22142, and the actual measured molecular weight is correct (as shown in Figure 10), without oxidation modification, and the product is relatively uniform.
  • Example 4 IFN- ⁇ (C166S) activity test after renaturation and purification
  • interferon can protect human amniotic membrane cells (WISH) from vesicular stomatitis virus ( The effect of VSV) destruction, the survival WISH cells are stained by the MTT method, and the absorbance is measured on a microplate reader to obtain the protective effect curve of the interferon on the WISH cells to determine the biological activity of the interferon.
  • WISH human amniotic membrane cells
  • MTT vesicular stomatitis virus
  • the absorbance is measured on a microplate reader to obtain the protective effect curve of the interferon on the WISH cells to determine the biological activity of the interferon.
  • the highest concentration of IFN- ⁇ (C166S) used is 10000ng/mL, 5 times of gradient dilution, a total of 10 dilutions.
  • the results are shown in Figure 11.
  • the IFN- ⁇ (C166S) sample played a significant role in protecting WISH cells from vesicular stomatitis virus (VSV).
  • the IFN- ⁇ (C166S) sample started to have an effect at a dose of 5ng/ml.
  • the protective effect on WISH cells is equivalent to the positive control IFN ⁇ at the doses that each exert the maximum function.

Abstract

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。

Description

干扰素-κ突变体及其制备方法 技术领域
本发明属于分子生物学领域，具体涉及一种干扰素-κ突变体及其制备方法。
背景技术
干扰素(interferon，IFN)，是由科学家Isaacs和Lindenmann于1957年发现流感病毒感染的鸡胚能分泌的一种抗流感病毒因子(Isaacs等，1957)，是一类具有广泛生物学活性的蛋白质，是可由多种细胞合成、分泌的重要细胞因子，具有抗病毒、抗肿瘤和免疫调节等多种作用(Takaoka A等，2003)，是机体天然免疫防御系统的重要组成部分。
干扰素属诱生蛋白，正常细胞一般不自发产生，属于发挥病毒防御功能最快的免疫体系。干扰素抗病毒效应往往早于特异的机体免疫反应，可有效地限制病毒复制，并具有抑制肿瘤细胞增殖和调节免疫功能的活性(LaFleur等，2001年；Nardelli等，2002年)，是世界范围内许可的治疗多种病毒性疾病、肿瘤和免疫紊乱的药物。目前的抗病毒药物只有单纯的抗病毒作用，而IFN则同时具备抗病毒和调节免疫功能的双重作用，所以抗病毒效果比一般药物强而持久。目前，在我国临床上广泛使用干扰素来治疗慢性乙型肝炎和慢性丙型肝炎等。
根据来源和结构，可将干扰素分为两大类Ⅰ型IFN和Ⅱ型IFN：Ⅰ型IFN包括IFN-α、IFN-β、IFN-κ、IFN-ε、IFN-ω、IFN-δ等13种亚型，Ⅱ型IFN只有IFN-γ一种(Bach EA等，1997年)。近年来，有新发现的具有干扰素活性的IFN-λ1(IL-28A)、IFN-λ2(IL-28B)、IFN-λ3(IL-29)(Kotenko S V等，2003年)，但它们结合的受体与Ⅰ型干扰素受体不同，故将其命名为Ⅲ型干扰素。
干扰素-κ(interferon kappa，IFN-κ)是近来发现的Ⅰ型干扰素家族成员，由207个氨基酸组成，其中包括27个氨基酸的信号肽，IFN-κ与其他Ⅰ型干扰素具有30％的同源性，由角质形成细胞、单核细胞和树突状细胞(DC)表达，对角质形成细胞和特定的淋巴样细胞群表现出紧密的向性(DeCarlo等人，2010年；LaFleur等人，2001年)。IFN-κ可通过与其它I型干扰素相同的信号通路，即与IFN受体(IFNR)1/2相互作用，激活抗病毒因子的表达。这些基因调节广泛的细胞反应，包括抗病毒效应，抗肿瘤效应，增强NK细胞活性以及激活适应性免疫反应。研究表明，在病毒感染后，IFN-κ选择性地表达在上皮角质细胞中，从而抑制脑心肌炎病毒(ECMV)和人类乳头瘤病毒(HPV)的复制(DeCarlo CA等，2010年)。此外IFN-κ还可抑制多种囊膜病毒的复制，包括流感病毒、寨卡病毒等，显示出广谱 抗病毒效果。
2020年1月上海市公共卫生临床中心提出的抗病毒喷剂其中一种主要成分即为IFN-κ，从实验室数据上看，新型冠状病毒(SARS-CoV-2，2019)对于干扰素比流感病毒和寨卡病毒对干扰素更敏感，因此喷剂对冠状病毒可能更有效。因此，IFN-κ已显示出与其他I型IFN相似的抗病毒活性，但它与其他I型IFN截然不同，因为它似乎以离散的自分泌而非旁分泌的细胞相关方式发出信号(LaFleur等，2001年)。在当前的IFN治疗方案中，大量副作用可能会限制IFN的治疗潜力，但是由于IFN-κ独特的分泌性质以及调节免疫分子的作用，可能会使目前的IFN治疗方案有所改善，因此具有更好的使用前景。但是由于其分子的性质原因，规模产业化可能是一个很大的挑战，所以有必要对其进行优化。
发明内容
有鉴于现有技术中的IFN-κ由于分子的性质原因，产量不高、变性和复性有难度、产物不均一等，难以进行规模产业化，本发明提供了一种IFN-κ突变体及其制备方法。本发明也可以只解决上述问题中的一种或几种。
本发明的一个方面提供了一种干扰素-κ突变体，在一个具体实施方式中，该干扰素-κ突变体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。
进一步地，将自由的半胱氨酸残基突变为侧链较小的氨基酸；将能够形成氧化修饰的甲硫氨酸残基部分或全部突变为侧链较小的氨基酸和/或疏水性氨基酸中的一种或多种。
进一步地，侧链较小的氨基酸为丝氨酸、丙氨酸或甘氨酸；疏水性氨基酸为缬氨酸、亮氨酸或异亮氨酸。
在一个实施方式中，干扰素-κ突变体还在野生型干扰素-κ的基础上，在N末端添加一个或多个氨基酸。
进一步地，在N末端添加的一个或多个氨基酸为侧链较小的氨基酸。
进一步地，侧链较小的氨基酸为丝氨酸、丙氨酸或甘氨酸。
可选地，野生型干扰素-κ的氨基酸序列如SEQ ID NO.1所示、或者包含如SEQ ID NO.1所示的氨基酸序列、或者包含与SEQ ID NO.1至少80％、90％、95％、98％、99％同一性的氨基酸序列。
进一步地，自由的半胱氨酸残基为SEQ ID NO.1所示的序列的第166位半胱氨酸残基；能够形成氧化修饰的甲硫氨酸残基为SEQ ID NO.1所示的序列的第52位、第112位、第115位和第120位甲硫氨酸残基。
在一个实施方式中，干扰素-κ突变体的氨基酸序列如下所述：
1)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸；即IFN-κ(C166S/G/A)；
2)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸，并在N末端添加一个丝氨酸、丙氨酸或甘氨酸；即IFN-κ(-1S/G/A，C166S/G/A)；
3)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸，在N末端添加一个丝氨酸、丙氨酸或甘氨酸，并将第112位甲硫氨酸残基和第115位甲硫氨酸残基替换为丝氨酸、丙氨酸、甘氨酸或疏水性氨基酸；即IFN-κ(-1S/G/A,M112*,M115*,C166S/G/A)；
4)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸，在N末端添加一个丝氨酸、丙氨酸或甘氨酸，并将第115位甲硫氨酸残基替换为丝氨酸、丙氨酸、甘氨酸或疏水性氨基酸；即IFN-κ(-1S/G/A,M115*,C166S/G/A)；
5)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸，在N末端添加一个丝氨酸、丙氨酸或甘氨酸，并将第115位甲硫氨酸残基和第120位甲硫氨酸残基替换为丝氨酸、丙氨酸、甘氨酸或疏水性氨基酸；即IFN-κ(-1S/G/A,C166S/G/A,M115*,M120*)；
6)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸，在N末端添加一个丝氨酸、丙氨酸或甘氨酸，并将第52位甲硫氨酸残基、第115位甲硫氨酸残基和第120位甲硫氨酸残基替换为丝氨酸、丙氨酸、甘氨酸或疏水性氨基酸；即IFN-κ(-1S/G/A,C166S/G/A,M52*,M115*,M120*)；
7)包含如1)～6)中任一个所示的氨基酸序列；或者
8)包含与1)～6)中任一个所示的氨基酸序列至少80％、90％、95％、98％、99％同一性的氨基酸序列。
本发明的第二个方面提供了一种分离的多核苷酸，在一个具体实施方式中，其编码如上所述的干扰素-κ突变体。
可选地，多核苷酸的序列如SEQ ID NO.4或6所示、或者包含如SEQ ID NO.4或6所示的多核苷酸序列、或者包含与SEQ ID NO.4或6的多核苷酸序列至少80％、90％、95％、98％、99％同一性的多核苷酸序列。
本发明的第三个方面提供了一种表达载体，在一个具体实施方式中，包含如上所述的一种分离的多核苷酸。
本发明的第四个方面提供了一种宿主细胞，在一个具体实施方式中，包含如上所述的一种分离的多核苷酸或如上所述的一种表达载体。
本发明的第五个方面提供了一种干扰素-κ突变体的制备方法，在一个具体实 施方式中，干扰素-κ突变体上所述；制备方法为：在表达系统中表达编码如上所述的干扰素-κ突变体的多核苷酸序列，或者利用如上所述表达载体进行表达，或者通过如上所述的宿主细胞表达。
可选地，表达系统为原核表达系统。
可选地，多核苷酸序列连接到表达载体pET21b或pET41a上，并在大肠杆菌BL21(DE3)中进行表达。
进一步地，制备方法还包括：将表达获得干扰素-κ突变体的包涵体进行体外复性和纯化。
可选地，体外复性为使用盐酸胍和/或DTT进行包涵体溶解，并在复性液中进行复性；纯化为离子交换层析纯化和/或反向层析纯化。
本发明的第五个方面提供了干扰素-κ突变体用于制备治疗疾病的药物或制剂中的用途，干扰素-κ突变体如上所述，或者通过如上所述的制备方法制备获得。
本发明的具体实施方式中的干扰素-κ突变体具有如下优势：
1)将野生型干扰素-κ中自由的半胱氨酸突变为其他氨基酸残基，比如侧链较小的氨基酸残基，有助于表达蛋白的体外复性和纯化。
2)将野生型干扰素-κ中能够形成氧化修饰的甲硫氨酸残基部分或全部突变为其他氨基酸残基，比如侧链较小的氨基酸残基和/或疏水性氨基酸，防止或减少在后续的体外复性和纯化中，甲硫氨酸形成氧化修饰(oxidation)造成的产物不均一的现象。
3)当使用大肠杆菌进行蛋白表达时，起始密码子生成的甲硫氨酸残基不会或难以掉落，会导致在后续的体外复性和纯化中，甲硫氨酸形成氧化修饰(oxidation)造成的产物不均一的现象。因此，在起始密码子之后，在野生型干扰素-κ的第一位亮氨酸残基之前(N末端)增加一个或多个氨基酸残基，以帮助起始密码子生成的甲硫氨酸残基的掉落，进一步避免产物不均一的现象。
4)本发明的具体实施方式中的干扰素，其对WISH细胞免受水泡性口炎病毒(VSV)破坏的作用效果与IFNβ类似。
附图说明
图1是本发明实施例1中IFN-κ(C166S)的PCR产物电泳图。其中，M为DNA分子量梯度；泳道1和泳道2分别为第一轮PCR和第二轮PCR产物。
图2是本发明实施例1中IFN-κ(-1S,M112A,M115A,C166S)的PCR产物电泳图。其中，M为Marker；泳道1和泳道2分别为第一轮PCR和第二轮PCR产物。
图3是本发明实施例2中IFN-κ(wt)蛋白在大肠杆菌中表达的SDS-PAGE电泳图。其中，M为蛋白标准；T为全菌蛋白；S为细菌裂解上清；P为包涵体。
图4是本发明实施例2中IFN-κ(C166S)蛋白在大肠杆菌中表达的SDS-PAGE 电泳图。其中，M为蛋白标准；T为全菌蛋白；S为细菌裂解上清；P为包涵体。
图5是本发明实施例2中IFN-κ(-1S,M112A,M115A,C166S)蛋白在大肠杆菌中表达的SDS-PAGE电泳图。其中，M为蛋白标准；T为全菌蛋白；S为细菌裂解上清；P为包涵体。
图6是本发明实施例3中IFN-κ(wt)和IFN-κ(C166S)包涵体复性后的SDS-PAGE电泳图，采用15％梯度胶。其中，M为蛋白标准(单位kD)，还原代表上样缓冲液中有还原剂，非还原代表不含还原剂。
图7是本发明实施例3中IFN-κ(C166S)包涵体复性及纯化后的SDS-PAGE电泳图，采用4～15％梯度胶。其中，M为蛋白标准(单位kD)，还原代表上样缓冲液中有还原剂，非还原代表不含还原剂。
图8是本发明实施例3中IFN-κ(-1S,M112A,M115A,C166S)包涵体复性及纯化后的SDS-PAGE电泳图，采用15％胶。其中，M为蛋白标准(单位kD)，还原代表上样缓冲液中有还原剂，非还原代表不含还原剂。
图9是采用LC-MS鉴定IFN-κ(C166S)的结果图。
图10是采用LC-MS鉴定IFN-κ(-1S,M112A,M115A,C166S)的结果图。
图11是IFN-κ(C166S)对WISH细胞保护作用的测定；其中，阳性对照为IFNβ，阴性对照为与I型干扰素不相关的蛋白样品。
具体实施方式
以下将结合实施例对本发明作进一步地说明，应理解这些实施例仅作为例证的目的，不用于限制本发明的保护范围。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。所采用的试剂，若无特殊说明，均为市售或公开渠道可以获得的试剂。
下文中，除另有说明，野生型干扰素-κ的氨基酸序列如SEQ ID NO.1所示，其是以NCBI中protein_id：NP_064509的氨基酸序列去除1～27位信号肽之后形成的。
除另有说明，干扰素-κ突变体的氨基酸序列中氨基酸位置，以SEQ ID NO.1所示的氨基酸序列为基准进行计算，即SEQ ID NO.1第一位亮氨酸的位置为1，第一位亮氨酸之后的氨基酸顺序计算位置；若干扰素-κ突变体在第一位亮氨酸之前(N末端)添加其他氨基酸，则添加的其他氨基酸的位置其位置按-1、-2依次计算(最靠近第一位亮氨酸的位置为-1)。
“自由的半胱氨酸残基”是指在干扰素-κ中不形成二硫键的半胱氨酸残基。
“能够形成氧化修饰的甲硫氨酸残基”是指根据空间构象和表面氨基酸分析， 可能暴露在蛋白表面的甲硫氨酸残基。
在氨基酸侧链的相对尺寸在本领域是熟知的，并且可以使用任何已知的度量标准比较，所述度量标准包括立体效应、电子密度等。以渐增的尺寸的顺序列出的氨基酸的一个实例是G、A、S、C、V、T、P、I、L、D、N、E、Q、M、K、H、F、Y、R、W。
疏水性氨基酸在本领域是熟知的，例如V、L、I。
本发明的一个具体实施方式中的干扰素-κ突变体，是通过在野生型干扰素-κ(可以是如SEQ ID NO.1所示的氨基酸序列，或者包含如SEQ ID NO.1所示的氨基酸序列、或者包含与SEQ ID NO.1至少80％、90％、95％、98％、99％同一性的氨基酸序列)的基础上，通过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形成的。在一些实施方式中，将自由的半胱氨酸残基突变为侧链较小的氨基酸；将能够形成氧化修饰的甲硫氨酸残基部分或全部突变为侧链较小的氨基酸和/或疏水性氨基酸。侧链较小的氨基酸包括但不限于，甘氨酸、丙氨酸、丝氨酸、缬氨酸、苏氨酸。疏水性氨基酸包括但不限于缬氨酸、亮氨酸、异亮氨酸。将自由的半胱氨酸残基突变为侧链较小的氨基酸残基，有助于后续表达蛋白的体外复性和纯化。将能够形成氧化修饰的甲硫氨酸残基部分或全部突变为侧链较小的氨基酸残基和/或疏水性氨基酸，可以防止或减少在后续的体外复性和纯化中，甲硫氨酸形成氧化修饰(oxidation)造成的产物不均一的现象。
在一个具体实施方式中，自由的半胱氨酸残基为SEQ ID NO.1所示的序列的第166位半胱氨酸残基；能够形成氧化修饰的甲硫氨酸残基为SEQ ID NO.1所示的序列的第52位、第112位、第115位和第120位甲硫氨酸残基。
在另一个具体实施方式中，干扰素-κ突变体可以通过表达系统进行表达。表达系统可以是原核表达系统，可以是真核表达系统或无细胞表达系统。当使用原核表达系统，比如大肠杆菌表达系统进行表达时，为了避免或减少起始密码子经翻译后形成的甲硫氨酸残基没有掉落，而在后续体外复性和纯化中该甲硫氨酸残基形成氧化修饰(oxidation)造成的产物不均一的现象，在起始密码子翻译形成的甲硫氨酸残基之后，添加一个或多个氨基酸残基，比如添加一个、两个或三个氨基酸残基，从而有助于甲硫氨酰氨基肽酶(MAP)催化对甲硫氨酸进行切除。添加的一个或多个氨基酸残基不是甲硫氨酸残基、不是半胱氨酸残基，可选地是侧链较小的氨基酸残基，包括但不限于，甘氨酸、丙氨酸、丝氨酸、缬氨酸、苏氨酸。
在另一个具体实施方式中，可以对野生型干扰素-κ(可以是如SEQ ID NO.1所示的氨基酸序列，或者包含如SEQ ID NO.1所示的氨基酸序列、或者包含与SEQ ID NO.1至少80％、90％、95％、98％、99％同一性的氨基酸序列)进行三种改进方式中的一种或两种或三种：1)将自由的半胱氨酸残基突变为半胱氨酸之外的氨 基酸，2)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形成的，3)在起始密码子翻译形成的甲硫氨酸残基之后，添加一个或多个氨基酸残基。
以下实施例中，野生型干扰素-κ的氨基酸序列如SEQ ID NO.1所示。
实施例1重组IFN-κ的设计及表达质粒的构建
IFN-κwt即野生型干扰素-κ，其氨基酸序列如SEQ ID NO.1所示，其核苷酸序列如SEQ ID NO.2所示。
为了有助于IFN-κ的体外复性和纯化和/或使产物单一，设计了如下的重组IFN-κ：
IFN-κ(-1S/G/A,C166S/G/A)，例如IFN-κ(-1S,C166S)；
IFN-κ(C166S/G/A)，例如IFN-κ(C166S)；
IFN-κ(-1S,M112*,M115*,C166S)，例如IFN-κ(-1S,M112A,M115A,C166S)、IFN-κ(-1S,M112V,M115A,C166S)；
IFN-κ(-1S,M115*,C166S)，例如IFN-κ(-1S,M115A,C166S)、IFN-κ(-1S,M115V,C166S)；
IFN-κ(-1S,C166S,M115*,M120*)，例如IFN-κ(-1S,C166S,M115A,M120A)、IFN-κ(-1S,C166S,M115A,M120L)；
IFN-κ(-1S,C166S,M52*,M115*,M120*)，例如IFN-κ(-1S,C166S,M52S,M115G,M120A)、IFN-κ(-1S,C166S,M52S,M115V,M120L)。
其中*代表侧链较小的氨基酸或疏水性氨基酸。
IFN-κ(C166S)即在野生型干扰素-κ的基础上，将第166位的半胱氨酸突变成丝氨酸，其氨基酸序列如SEQ ID NO.3所示，其核苷酸序列如SEQ ID NO.4所示。
IFN-κ(-1S,M112A,M115A,C166S)即在野生型干扰素-κ的基础上，在第1位亮氨酸之前(-1位)添加一个丝氨酸，将第112位和第115位的甲硫氨酸突变成丙氨酸，将第166位的半胱氨酸突变成丝氨酸，其氨基酸序列如SEQ ID NO.5所示，其核苷酸序列如SEQ ID NO.6所示。
1.1基因和引物合成
IFN-κwt的核苷酸序列(如SEQ ID NO.2所示)由中美泰和生物技术(北京)有限公司合成。
SEQ ID NO.4和SEQ ID NO.6的PCR引物由上海生工生物工程股份有限公司合成，具体如表1所示：
表1引物序列
Figure PCTCN2021090472-appb-000001
1.2表达质粒的构建
以SEQ ID NO.2基因为模板，进行2轮PCR扩增(分别以SEQ4-F1和SEQ4-R1、SEQ4-F1和SEQ4-R2为引物)，扩增获得的PCR产物进行DNA凝胶回收(如图1所示)，然后重组进pET21b质粒，构建出pET21b-IFNK C166S表达质粒；而以获得的pET21b-IFNK(C166S)表达质粒为模板，进行2轮PCR扩增(分别以SEQ6-F1和SEQ6-R1、SEQ6-F2和SEQ6-R2为引物)，扩增获得的PCR产物进行DNA凝胶回收(如图2所示)，构建出pET41a-IFNK(-1S,M112A,M115A,C166S)表达质粒。以上均按照《分子克隆》进行操作。
同时构建了pET21b-IFNK(wt)表达质粒。
实施例2IFN-κwt、重组IFN-κ(C166S)和IFN-κ(-1S,M112A,M115A,C166S)在大肠杆菌中的表达
取1μL实施例1中制备的表达质粒转化表达菌BL21(DE3)，挑取1个单菌落进行保菌。
按1：2000比例进行接种，次日按1：50比例转接到含Amp抗性的500mL LB培养基的三角瓶中，37℃，220rpm，培养至OD 600＝2，加入终浓度为0.5mM IPTG，37度诱导3h后收菌。收获的菌体用缓冲液进行悬浮，并进行高压破碎， 800bar，3次，然后将表达的蛋白进行SDS-PAGE还原性电泳检测，查看是否生成正确大小的蛋白。以上均按照《分子克隆》进行操作。
结果如图3、图4和图5所示，IFN-κwt，IFN-κ(C166S)和IFN-κ(-1S,M112A,M115A,C166S)均获得了在20kD～25kD之间有特异蛋白被表达，且目标蛋白主要以包涵体的形式表达。
实施例3重组IFN-κwt，IFN-κ(C166S)和IFN-κ(-1S,M112A,M115A,C166S)体外复性和纯化
体外复性和纯化中使用的包涵体，采用如实施例2中的培养、诱导及破碎方法获得。
3.1包涵体溶解和复性
用6M盐酸胍和20mM DTT分别溶解实施例2中得到的含有目的蛋白的包涵体，然后分别将溶解后的包涵体按1:100的比例稀释到复性液中(20mM Tris-HCl，1mM半胱氨酸(cysteine)，1mM胱氨酸(cystine)，0.1％SDS，pH9.0)，室温过夜，作为复性样品，即IFN-κwt，IFN-κ(C166S)或IFN-κ(-1S,M112A,M115A,C166S)的复性样品。
如图6所示，IFN-κ(C166S)在还原和非还原胶上都能看到特异性目的条带，且折叠好的目的蛋白在非还原条件下要比还原条件下迁移的快；而IFN-κwt只在还原条件下看到目的条带，而非还原则没有，可能是形成了沉淀或是聚合物。
3.2离子交换层析纯化
复性后的样品经过10000g，30分钟离心，上清液上样20ml的阳离子交换柱子(购自GE healthcare)，然后进行NaCl梯度洗脱，根据SDS-PAGE选择目的蛋白所在的收集管合并保存，此步可以捕获目的蛋白并去除一些杂蛋白。
3.3反相层析纯化
离子交换层析后合并的蛋白再经过反相层析柱子C8(购自YMC)，采用乙腈梯度洗脱，去除折叠错误和一些修饰的蛋白。
3.4离子交换精细纯化
反相层析柱子C8后合并的蛋白适当稀释后进行阳离子交换精细纯化，采用NaCL梯度洗脱，去除一些杂蛋白。
3.5脱盐柱脱盐换液
采用脱盐柱G25(购自GE healthcare)对IFN-κ蛋白进行脱盐换液处理，最终缓冲液为PBS，pH 7.4。
最终样品SDS-PAGE电泳结果如图7和图8显示，为单一条带，且折叠好的目的蛋白在非还原条件下要比还原条件下迁移的快。
3.6 LC-MS鉴定
IFN-κ(C166S)的理论分子量为22306(带有起始Met)，而经过LC-MS鉴定(正离子模式，cone voltage：70V；Capillary voltage：1.5kV；mass range:400-7000m/z)，本实施例中的IFN-κ(C166S)分子量正确(如图9)，复性成功，但是也有连续的+16的分子量，提示为氧化修饰(oxidation)，造成产物的不均一；而经过突变后的IFN-κ(-1S,M112A,M115A,C166S)的理论分子量为22142，实际测得分子量正确(如图10)，且没有氧化修饰，产物较均一。
实施例4复性纯化后的IFN-κ(C166S)活性测试
本发明中利用2015年版《中国药典》三部通则3523干扰素生物学活性测定法来检测干扰素的活性，本法系依据干扰素可以保护人羊膜细胞(WISH)免受水泡性口炎病毒(VSV)破坏的作用，用MTT法对存活的WISH细胞染色，在酶标仪测定其吸光度，可得到干扰素对WISH细胞的保护效应曲线，以此测定干扰素生物学活性。其中，使用到IFN-κ(C166S)的最高浓度为10000ng/mL，5倍梯度倍比稀释，共10个稀释度。
结果如图11所示，IFN-κ(C166S)样品起到了明显保护WISH细胞免受水泡性口炎病毒(VSV)破坏的作用，IFN-κ(C166S)样品从5ng/ml的剂量开始发生效果，并且在各自发挥最大功能的剂量时，对WISH细胞的保护效果与阳性对照IFNβ相当。

Claims (19)

  1. 干扰素-κ突变体，其特征在于，所述干扰素-κ突变体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。
  2. 如权利要求1所述的干扰素-κ突变体，其特征在于，将所述自由的半胱氨酸残基突变为侧链较小的氨基酸；将所述能够形成氧化修饰的甲硫氨酸残基部分或全部突变为侧链较小的氨基酸和/或疏水性氨基酸中的一种或多种。
  3. 如权利要求2所述的干扰素-κ突变体，其特征在于，所述侧链较小的氨基酸为丝氨酸、丙氨酸或甘氨酸；所述疏水性氨基酸为缬氨酸、亮氨酸或异亮氨酸。
  4. 如权利要求1所述的干扰素-κ突变体，其特征在于，所述干扰素-κ突变体还在野生型干扰素-κ的基础上，在N末端添加一个或多个氨基酸。
  5. 如权利要求4所述的干扰素-κ突变体，其特征在于，在N末端添加的一个或多个氨基酸为侧链较小的氨基酸。
  6. 如权利要求5所述的干扰素-κ突变体，其特征在于，所述侧链较小的氨基酸为丝氨酸、丙氨酸或甘氨酸。
  7. 如权利要求1所述的干扰素-κ突变体，其特征在于，所述野生型干扰素-κ的氨基酸序列如SEQ ID NO.1所示、或者包含如SEQ ID NO.1所示的氨基酸序列、或者包含与SEQ ID NO.1至少80％、90％、95％、98％、99％同一性的氨基酸序列。
  8. 如权利要求7所述的干扰素-κ突变体，其特征在于，所述自由的半胱氨酸残基为SEQ ID NO.1所示的序列的第166位半胱氨酸残基；所述能够形成氧化修饰的甲硫氨酸残基为SEQ ID NO.1所示的序列的第52位、第112位、第115位和第120位甲硫氨酸残基。
  9. 如权利要求8所述的干扰素-κ突变体，其特征在于，所述干扰素-κ突变体的氨基酸序列如下所述：
    1)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸；
    2)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸，并在N末端添加一个丝氨酸、丙氨酸或甘氨酸；
    3)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸，在N末端添加一个丝氨酸、丙氨酸或甘氨酸，并将第112位甲硫氨酸残基和第115位甲硫氨酸残基替换为丝氨酸、丙氨酸、甘氨酸或疏水性氨基酸；
    4)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸，在N末端添加一个丝氨酸、丙氨酸或甘氨酸，并将第115位甲硫氨酸残基替换为丝氨酸、丙氨酸、甘氨酸或疏水性氨基酸；
    5)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸，在N末端添加一个丝氨酸、丙氨酸或甘氨酸，并将第115位甲硫氨酸残基和第120位甲硫氨酸残基替换为丝氨酸、丙氨酸、甘氨酸或疏水性氨基酸；
    6)在SEQ ID NO.1所示的氨基酸序列基础上，将第166位半胱氨酸残基替换为丝氨酸、丙氨酸或甘氨酸，在N末端添加一个丝氨酸、丙氨酸或甘氨酸，并将第52位甲硫氨酸残基、第115位甲硫氨酸残基和第120位甲硫氨酸残基替换为丝氨酸、丙氨酸、甘氨酸或疏水性氨基酸；
    7)包含如1)～6)中任一个所示的氨基酸序列；或者
    8)包含与1)～6)中任一个所示的氨基酸序列至少80％、90％、95％、98％、99％同一性的氨基酸序列。
  10. 一种分离的多核苷酸，其特征在于，其编码如权利要求1～9中任一项所述的干扰素-κ突变体。
  11. 如权利要求10所述的分离的多核苷酸，其特征在于，所述多核苷酸的序列如SEQ ID NO.4或6所示、或者包含如SEQ ID NO.4或6所示的多核苷酸序列、或者包含与SEQ ID NO.4或6的多核苷酸序列至少80％、90％、95％、98％、99％同一性的多核苷酸序列。
  12. 一种表达载体，其特征在于，包含如权利要求10或11所述的一种分离的多核苷酸。
  13. 一种宿主细胞，其特征在于，包含如权利要求10或11所述的一种分离的多核苷酸或如权利要求12所述的一种表达载体。
  14. 一种干扰素-κ突变体的制备方法，其特征在于，所述干扰素-κ突变体如权利要求1～9中任一项所述；所述制备方法为：在表达系统中表达编码如权利要求1～9中任一项所述的干扰素-κ突变体的多核苷酸序列，或者利用如权利要求12所述表达载体进行表达，或者通过如权利要求13所述的宿主细胞表达。
  15. 如权利要求14所述的干扰素-κ突变体的制备方法，其特征在于，所述表达系统为原核表达系统。
  16. 如权利要求15所述的干扰素-κ突变体的制备方法，其特征在于，所述多核苷酸序列连接到表达载体pET21b或pET41a上，并在大肠杆菌BL21(DE3)中进行表达。
  17. 如权利要求16所述的干扰素-κ突变体的制备方法，其特征在于，所述制备方法还包括：将表达获得干扰素-κ突变体的包涵体进行体外复性和纯化。
  18. 如权利要求17所述的干扰素-κ突变体的制备方法，其特征在于，所述体外复性为使用盐酸胍和/或DTT进行包涵体溶解，并在复性液中进行复性；所述纯化为离子交换层析纯化和/或反向层析纯化。
  19. 干扰素-κ突变体用于制备治疗疾病的药物或制剂中的用途，其特征在于，所述干扰素-κ突变体如权利要求1～9中任一项所述，或者通过如权利要求14～18中任一项所述的制备方法制备获得。
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EP0260350A1 (en) * 1986-09-05 1988-03-23 Cetus Oncology Corporation Oxidation-resistant interferon-beta muteins and their production; formulations containing such muteins
US20040137581A1 (en) * 2002-10-01 2004-07-15 Xencor Interferon variants with improved properties
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EP0260350A1 (en) * 1986-09-05 1988-03-23 Cetus Oncology Corporation Oxidation-resistant interferon-beta muteins and their production; formulations containing such muteins
US20040137581A1 (en) * 2002-10-01 2004-07-15 Xencor Interferon variants with improved properties
CN112521480A (zh) * 2020-12-25 2021-03-19 山东晶辉生物技术有限公司 一种人干扰素-κ突变体及其制备方法

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