Method for improving expression quantity of mouse interferon protein

Abstract

The invention provides a method for improving the expression quantity of mouse interferon protein. Specifically, the method comprises the step of optimizing a coding nucleotide sequence of the IFN-gamma protein, so that the recombinant IFN-gamma protein is expressed in vitro. According to the optimized nucleotide coding sequence, the free energy of an mRNA secondary structure of the nucleotide coding sequence is reduced, the in-vitro expression quantity of IFN-gamma protein is greatly improved and reaches up to 75mg / L, and the activity of recombinant protein is improved.

Description

Technical Field

[0001] This invention belongs to the technical field, and more specifically, this invention relates to a method for increasing the expression level of mouse-derived interferon protein. Background Technology

[0002] Type II interferon (IFN-γ) is mainly produced by immune cells such as T cells and NK cells, playing a central role in antibacterial, antiviral, and antitumor immunity. Its function is primarily achieved through the JAK-STAT signaling pathway: after IFN-γ binds to its receptor IFNGR1, it triggers a conformational change in its intracellular domains, thereby recruiting and activating JAK1, JAK2 kinases, and the STAT1 transcription factor. Once activated STAT1 enters the nucleus, it initiates the transcription of a series of interferon-regulated genes, including IRF1, creating a cascade amplification effect.

[0003] In antigen presentation, IFN-γ optimizes the proteolytic process by inducing the replacement of catalytic proteasome subunits with immunoproteasome subunits and upregulating the expression of proteasome activator PA28, thereby increasing the diversity, quality, and efficiency of MHC class I molecule-presented peptides. Simultaneously, it significantly enhances the expression levels of MHC class II molecules on the cell surface by promoting the expression of cathepsins B, H, and L. Furthermore, IFN-γ participates in regulating the development, quiescence, and differentiation of hematopoietic stem cells, profoundly influencing immune homeostasis.

[0004] Existing IFN-γ purification techniques mainly include:

[0005] (1) IFN-γ was expressed and purified using the E. coli system;

[0006] (2) Improve protein activity through refolding.

[0007] Disadvantages of existing technology:

[0008] (1) The protein expression level is not high;

[0009] (2) A complex operation is required.

[0010] Therefore, there is an urgent need in this field to develop a simple method to increase the expression level of IFN-γ protein. Summary of the Invention

[0011] The purpose of this invention is to provide a method for preparing soluble IFN-γ protein.

[0012] In a first aspect of the invention, an optimized polynucleotide encoding a soluble IFN-γ recombinant protein is provided, the polynucleotide having the nucleotide sequence shown in SEQ ID NO.4, or having at least 90% sequence identity with SEQ ID NO.4.

[0013] In another preferred embodiment, the nucleotide sequence of the polynucleotide has at least 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO.4.

[0014] In another preferred embodiment, the polynucleotide encodes the IFN-γ protein shown in SEQ ID NO.1.

[0015] In another preferred embodiment, the polynucleotide encodes a mouse-derived IFN-γ protein.

[0016] In another preferred embodiment, the homology between SEQ ID NO.4 and the original nucleotide sequence of the IFN-γ protein is 77.19%.

[0017] In another preferred embodiment, the polynucleotide is codon-optimized based on the original nucleic acid sequence.

[0018] In another preferred embodiment, the polynucleotide optimizes the secondary structure of the mRNA it encodes.

[0019] In another preferred embodiment, the free energy of the secondary structure of the mRNA encoded by the polynucleotide is lower than the free energy of the secondary structure of the mRNA encoded by the original nucleotide sequence of the IFN-γ protein.

[0020] In another preferred embodiment, the free energy of the secondary structure of the mRNA encoded by the polynucleotide is -92.60 kcal / mol.

[0021] In another preferred embodiment, the minimum free energy of the mRNA secondary structure encoded by the original nucleotide sequence is -71.90 kcal / mol.

[0022] In another preferred embodiment, the polynucleotide eliminates hidden splicing sites in the original nucleic acid sequence.

[0023] In a second aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide described in the first aspect of the invention.

[0024] In another preferred embodiment, the vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, and retroviruses.

[0025] In another preferred embodiment, the expression vector is selected from pet28a, pET-32a, pCold series, and pSMART series.

[0026] In another preferred embodiment, the expression vector further includes a protein purification tag selected from GST, SUMO, His, and FLAG.

[0027] In a third aspect of the invention, a host cell is provided, the host cell containing the vector described in the second aspect of the invention, or having the polynucleotides described in the first aspect of the invention integrated into its genome.

[0028] In another preferred embodiment, the host cell is selected from Escherichia coli, CHO, HEK-293, Sf9, and Pichia pastoris.

[0029] In a fourth aspect of the invention, a method for preparing soluble IFN-γ protein is provided, the method comprising the steps of:

[0030] (a) The host cells of the third aspect of the present invention are cultured under suitable culture conditions to obtain a culture; and

[0031] (b) Soluble IFN-γ protein was isolated and purified from the culture.

[0032] In a fifth aspect of the invention, an IFN-γ recombinant protein is provided, characterized in that the recombinant protein is produced by the cells described in the third aspect of the invention, or prepared by the method described in the fourth aspect of the invention.

[0033] In another preferred embodiment, the expression level of the recombinant IFN-γ protein is as high as 75 mg / L.

[0034] In a sixth aspect of the invention, the use of a polynucleotide as described in the first aspect of the invention, an expression vector as described in the second aspect of the invention, a host cell as described in the third aspect of the invention, or a recombinant protein as described in the fifth aspect of the invention, for the preparation of formulations or compositions is provided.

[0035] In another preferred embodiment, the formulation is a laboratory formulation.

[0036] In another preferred embodiment, the composition is a pharmaceutical composition.

[0037] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0038] Figure 1 The secondary structure of the mRNA encoded by the original nucleic acid sequence of the mouse IFN-γ protein is shown.

[0039] Figure 2 The homology comparison between the first optimized nucleic acid sequence and the original mouse IFN-γ nucleic acid sequence is shown.

[0040] Figure 3The secondary structure of the mRNA encoded by the second optimized nucleic acid sequence is shown.

[0041] Figure 4 The homology comparison between the second optimized nucleic acid sequence and the original mouse IFN-γ nucleic acid sequence is shown.

[0042] Figure 5 The expression of IFN-γ protein without sequence optimization is shown. (1: Whole bacteria; 2: Precipitate; 3: Supernatant; 4: Triton 114 supernatant; 5: Triton 114 precipitate (IBS))

[0043] Figure 6 The expression of IFN-γ protein after the second sequence optimization is shown. (Whole: whole bacteria, M: marker, S: supernatant, P: precipitate, FT: flow-through, F0: elution, F100: 100mM NaCl elution, F200: 200mM NaCl elution, F300-1-2: 300mM NaCl elution, F500-1-2-3: 500mM NaCl elution, F1M: 1M NaCl elution, R: column residue)

[0044] Figure 7 The molecular sieve peak diagram of the IFN-γ protein after the second sequence optimization is shown.

[0045] Figure 8 The image shows the SDS-PAGE purity determination of the IFN-γ protein after molecular sieving following the second sequence optimization. (M represents the marker, S represents the protein before loading, A9, A11, B12, B10, B8, B6, B4, and B2 represent proteins collected in tubes at different elution positions, and B12NR represents the protein in tube B12 under non-reducing conditions.)

[0046] Figure 9 The results show the activity assay of mouse IFN-γ protein recombinantly expressed in Escherichia coli after the second optimization of the nucleic acid sequence.

[0047] Figure 10 The activity assay results of mouse IFN-γ protein recombinantly expressed in Escherichia coli with unoptimized nucleic acid sequences are shown. Detailed Implementation

[0048] Through extensive and in-depth research, the inventors have developed an optimized polynucleotide encoding a soluble IFN-γ recombinant protein. Compared to the original nucleotide sequence of the IFN-γ protein, this polynucleotide reduces the free energy of the mRNA's secondary structure, enabling high expression of the IFN-γ recombinant protein in *E. coli* in vitro, reaching an expression level of up to 75 mg / L, accounting for 35% of the total bacterial protein, and existing in a soluble form, greatly simplifying the protein purification process. This invention is based on this foundation.

[0049] the term

[0050] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0051] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0052] Codon optimization

[0053] Codon optimization refers to the technique of redesigning the nucleotide sequence of a target protein by replacing synonymous codons in its encoding gene without altering the target protein's amino acid sequence, thereby enabling efficient and stable expression in a specific host expression system. There are 64 codons corresponding to 20 amino acids and a stop signal. Except for methionine and tryptophan, other amino acids are encoded by multiple (2-6) codons. These different codons encoding the same amino acid are called "synonymous codons."

[0054] Although synonymous codons encode the same amino acids, different organisms (and even different tissues of the same organism) exhibit strong preferences for which codons to use. This preference is highly correlated with the abundance and distribution of intracellular tRNAs.

[0055] High-frequency codons correspond to high tRNA abundance, resulting in fast and accurate translation.

[0056] Rare codons correspond to low tRNA abundance, causing ribosomes to "get stuck" during translation, which may lead to decreased translation efficiency, increased translation errors, or even mRNA degradation or protein misfolding.

[0057] Codon optimization is not simply about replacing all codons with the one the host uses most often; it is a process of balancing multiple objectives.

[0058] The most common codon optimization method is to replace the codon for each amino acid with the most frequently used codon in the host genome, which maximizes the translation rate. However, overuse of a single optimal codon can lead to unfavorable secondary structures in mRNA or introduce unexpected regulatory sequences.

[0059] In addition, the following optimization methods can be considered:

[0060] mRNA secondary structure optimization:

[0061] The sequence was adjusted to weaken the stable secondary structures at the 5' end of the mRNA (especially near the start codon), making it easier for ribosomes to bind and initiate translation. Simultaneously, the structure of the entire coding region was optimized to avoid the formation of globally stable structures that could hinder ribosome movement.

[0062] GC content adjustment:

[0063] Adjusting the GC content of sequences to adapt them to the host genome. For example, mammalian genes have high GC content, while some bacteria (such as Bacillus subtilis) prefer AT enrichment. Appropriate GC content contributes to stable transcription and efficient translation.

[0064] Eliminate hidden splice sites, regulatory elements, and repetitive sequences:

[0065] In eukaryotic hosts (such as CHO cells), it is necessary to remove intron splicing sites that may be misidentified. This includes eliminating cryptic prokaryotic promoters, ribosome binding sites, or unstable repetitive sequences (such as polyA signals) that may affect transcription or translation.

[0066] In this invention, the codon optimization strategy for IFN-γ protein adopted mRNA secondary structure optimization combined with the elimination of hidden splicing sites. After multiple sequence optimizations, the optimal nucleotide sequence shown in SEQ ID NO.4 was unexpectedly obtained. This optimal nucleotide sequence greatly improved the expression level and protein activity of IFN-γ protein when recombinantly expressed in Escherichia coli.

[0067] IFN-γ protein

[0068] IFN-γ is a core multifunctional cytokine of the immune system. Its main functions are to activate macrophages, enhance antigen presentation, and coordinate Th1 immune responses, thereby playing a key role in combating intracellular pathogens, viral infections, and tumors.

[0069] In vivo, IFN-γ is mainly produced by three types of immune cells in response to stimulation by antigens (such as pathogens and tumor antigens) or certain cytokines (such as IL-12 and IL-18):

[0070] Activated T lymphocytes: such as CD4+Th1 cells: the primary source and key to adaptive immune responses; CD8+ cytotoxic T cells: generated when killing virus-infected cells or tumor cells; and γδ T cells: a bridge between innate and adaptive immunity.

[0071] Natural killer cells: In the early immune response, NK cells are an important source of innate immunity to IFN-γ.

[0072] NKT cells: They possess characteristics of both T cells and NK cells and can also rapidly produce IFN-γ.

[0073] When the original nucleic acid sequence of IFN-γ protein is recombinantly expressed in vitro, the protein expression level is low and it mainly exists in the form of inclusion bodies, requiring denaturation and renaturation before use. Therefore, this invention optimizes its original nucleic acid sequence. The optimized nucleic acid sequence reduces the free energy of its encoded mRNA, increases the protein expression level, and the protein obtained by recombinant expression is expressed in a soluble form with higher activity.

[0074] Preparation and purification of IFN-γ protein

[0075] The IFN-γ protein of this invention is a recombinant polypeptide produced from prokaryotic host cells (e.g., *Escherichia coli*) using recombinant technology. The polypeptide of this invention may or may not include an initial methionine residue.

[0076] Once the optimized coding sequence of this invention is obtained, the relevant sequence can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector (e.g., the pet28a vector), transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods.

[0077] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0078] The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0079] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or IFN-γ coding sequences, and methods for generating the polypeptides of the present invention via recombinant technology.

[0080] Using conventional recombinant DNA techniques, the polynucleotide sequence of this invention can be used to express or produce recombinant IFN-γ peptides. Generally, the following steps are involved:

[0081] (1). Transform or transduce suitable host cells using the polynucleotide encoding IFN-γ of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0082] (2) Host cells cultured in a suitable culture medium;

[0083] (3) Isolate and purify proteins from culture media or cells.

[0084] In this invention, the IFN-γ polynucleotide sequence can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0085] Methods well known to those skilled in the art can be used to construct expression vectors containing IFN-γ coding DNA sequences and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0086] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or tetracycline or ampicillin resistance.

[0087] In this invention, the *E. coli* recombinant expression system is preferred. Alternatively, the optimized nucleotide recombinant expression system of this invention can also be used to express the IFN-γ protein, such as the CHO, HEK-293, Sf9, and Pichia pastoris expression systems.

[0088] In this invention, the IFN-γ recombinant protein is a soluble protein secreted extracellularly. If desired, the recombinant protein can be separated and purified using various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: treatment with protein precipitants (salting out), centrifugation, permeation, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0089] Compared with the prior art, the main advantages of the present invention include:

[0090] 1. Compared with the original nucleic acid sequence of IFN-γ protein, the nucleic acid sequence optimized by this invention after multiple rounds of recombinant expression greatly improves the protein expression level (optimized 75 mg / L VS original 59 mg / L) and increases the proportion of the target protein in the whole bacterial protein (optimized 35% VS original 11%).

[0091] 2. Compared with the IFN-γ protein expressed by recombination of the original sequence, the IFN-γ protein expressed by recombination of the optimized nucleic acid sequence of the present invention has higher protein activity, and its EC50 for binding to CD119 is 82.44 ng / mL.

[0092] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0093] Experimental materials

[0094] SOB and LB media, kanamycin, IPTG (β-thiogalactoside), Tris (tris(hydroxymethyl)aminomethane), GuHCl (guanidine hydrochloride), TCEP (tris(2-carbonylethyl)phosphohydrochloride), ULP enzyme, imidazole, PBS (phosphate buffer).

[0095] Experimental methods

[0096] 1. Gene synthesis

[0097] The sequences found on Uniprot were designed and sent to a gene company for sequence optimization and synthesis. The gene was synthesized into the pet28a vector and recombinantly expressed in E. coli.

[0098] 2. Protein expression

[0099] 2.1 Plasmid dissolution

[0100] (1) The lyophilized plasmid was centrifuged at 12000 rpm for 1 min;

[0101] (2) Add sterile ultrapure water at a ratio of 1:25 (1 μg plasmid to 25 μL water) to dissolve the plasmid.

[0102] 2.2 Transformation

[0103] (1) Take 1 μL of plasmid and add it to 50 μL of competent cells, then incubate on ice for 30 min;

[0104] (2) Heat shock at 42℃ for 90 seconds (strictly control the time), then remove and place in an ice bath for 3 minutes;

[0105] (3) Add 600 μL of antibiotic-free LB liquid medium, incubate at 37℃ and 220 rpm for 1 h;

[0106] (4) Take 100 μL of activated bacterial cells and spread them evenly on the corresponding resistant LB plate. Incubate at 37°C upright for 15 min. After the liquid on the surface of the culture medium is absorbed, invert the plate and incubate overnight at 37°C.

[0107] 2.3 Preparation for Scale-up Culture

[0108] (1) 10x phosphate mother liquor: 0.17M KH2PO4, 0.72M K2HPO4;

[0109] (2) Ampicillin stock solution: 100 mg / ml; Kanamycin stock solution: 50 mg / ml

[0110] (3) IPTG stock solution: 1 mol / L

[0111] (4) TB basal culture medium: 12 g / L Tryptone, 24 g / L Yeast Extract, 4 ml / L Glycerol

[0112] 2.4 Scale-up culture

[0113] (1) Pick a round and plump single colony from the plate and put it into 25 mL of LB medium. Incubate at 37℃ and 220 rpm for 16 h.

[0114] (2) The next day, add 1 ml of the corresponding antibiotic stock solution and 100 ml of 10x phosphate stock solution to 1 L TB basal medium (3 L Erlenmeyer flask), mix well; add 25 mL of seed solution, incubate at 37℃ and 200 rpm for 5 h, then add 0.5 mL of IPTG stock solution, and adjust the temperature of the shaker to 16℃ and continue induction culture overnight.

[0115] (3) Stop shaking the bacteria before 9:00 a.m. the next day; centrifuge at 8000 rpm and 4°C for 5 min, collect the bacteria, and freeze at -80°C.

[0116] 2.5 Small-scale identification

[0117] (1) Centrifuge to collect bacterial pellet, add 10 mL of lysis buffer (PBS, pH 7.4), and sonicate (100 W, sonicate for 2 s, stop for 4 s, sonicate for 5 min);

[0118] (2) Take 20 μL of the suspension from the lysed suspension, add 20 μL of Loading Buffer to prepare a whole bacterial sample, and take 20 μL of the suspension; take 20 μL of the suspension from the lysed suspension, centrifuge at 12000 rpm for 2 min, discard the supernatant, add 20 μL of Loading Buffer to prepare a precipitate sample; take 20 μL of the suspension from the lysed suspension, centrifuge at 12000 rpm for 2 min, take 20 μL of the supernatant, add 20 μL of Loading Buffer to prepare a supernatant sample. The whole bacterial sample, supernatant, and precipitate sample are identified by SDS-PAGE electrophoresis.

[0119] 3. Protein purification

[0120] 3.1 Cell disruption

[0121] (1) Weigh the bacterial cells and add the disruption liquid at a mass-to-volume ratio of 1:10;

[0122] (2) Stir the bacterial solution in a homogenizer for 30s to 60s;

[0123] (3) Place the mixed bacterial solution in an ice-water mixture and sonicate for 20 minutes. Use an ultrasonic power of 500W, sonicate for 3 seconds and stop for 6 seconds. After sonication, observe whether the bacterial solution is uniform. If it is obviously lumpy, granular, or viscous, continue sonicating or crush and stir it before sonicating until the ultrasonic solution is uniform.

[0124] (4) After sonication, take 40 μL of the whole liquid and record it as the whole. Centrifuge the bacterial solution at 12000 rpm for 30 min and take 40 μL of the supernatant as the supernatant sample and record it as S. Prepare the sample at a volume ratio of 1:1. Resuspend the precipitated sample in 80 μL of loading buffer (containing DTT) and record it as P.

[0125] (5) After centrifugation, the sample is submitted for the next purification experiment.

[0126] 3.2 Ion Purification

[0127] Reagent formulation:

[0128] Buffer A: PB PH7.0

[0129] Buffer B: PB pH 7.0 1M NaCl

[0130] BufferC: PB PH7.0 0.5% TritonX114

[0131] Operating steps

[0132] (1) Take the empty column tube of the chromatography column soaked in alkaline solution and wash the inner wall of the tube with water free of endotoxin.

[0133] (2) Mix the materials evenly and take the required amount of filler;

[0134] (3) Rinse with 10 CV of endotoxin-free water;

[0135] (4) Wash with Buffer A for 10 CV;

[0136] (5) Load the supernatant sample onto the balanced packing material, recover the flow through, and record it as FT;

[0137] (6) After loading the sample, reequilibrate the column with Buffer A and collect the fraction as Wx;

[0138] (7) Rinse with Buffer C for 20 C v, and collect the fraction as F114-x;

[0139] (8) Rinse with Buffer A for 10 Cv;

[0140] (9) Prepare a 100mM-1M salt solution using BufferA and BufferB for elution. During elution, use Bradford spot until the solution no longer turns blue.

[0141] (10) Electrophoresis detection: Take the supernatant, flow through, add 40 μL each of 100 mM and 1 M elution buffer to 10 μL loading buffer (containing DTT), and record them as S, FT, F100 and F1M respectively, and run SDS-Page.

[0142] 3.3 Molecular sieve purification

[0143] (1) Regeneration: Rinse with 0.5M NaOH for 1 CV;

[0144] (2) Water washing: Rinse with ddH2O for 1 CV;

[0145] (3) Equilibration: Rinse with equilibration solution 1.2 CV;

[0146] (4) Sample loading: Load the sample directly using a loop metering loop or system pump, with the flow rate being the same as the equilibrium flow rate;

[0147] (5) Elution: Elute with the equilibration buffer for another 1 CV and collect the elution in separate tubes.

[0148] 4. Protein activity assay (ELISA)

[0149] Its binding ability was determined by functional ELISA. First, the test protein (Mouse IFN-γ) was directly coated onto the ELISA plate, followed by the addition of its potential receptor protein (Mouse CD119, i.e., IFN-γR1). The activity of IFN-γ was assessed by detecting the "signal" of CD119.

[0150] 4.1 Reagent Formulation

[0151] (1) Pre-cooled sterile water;

[0152] (2) Washing solution: 1XPBST;

[0153] (3) Blocking solution: PBST with 1% BSA;

[0154] (4) TMB colorimetric solution: Mix colorimetric solution A and colorimetric solution B in a 1:1 ratio before use, and prepare fresh before use;

[0155] (5) Termination solution (2M H2SO4).

[0156] 4.2 Operating Procedures

[0157] (1) Coating: First, dilute the protein with water to 200 μg / mL, then dilute the mouse IFN-γ protein with 1XPBS to 10 μg / mL, 100 μL / well, and incubate overnight at 4°C.

[0158] (2) Washing the plate: Wash the plate with washing solution, 260 μL / well, wash once, and pat dry;

[0159] (3) Blocking: Block with 1% BSA in 1*PBST (blocking solution), 150 μL / well, 25℃, 450 rpm for 4 h;

[0160] (4) First, dilute the protein with water to 200 μg / mL, then dilute Mouse CD119 to concentrations of 0, 0.01, 0.1, 1, 5, 10, 50, 100, 500, 1000, 2000, 5000, and 10000 ng / mL.

[0161] (5) Add Mouse CD119 protein: 100 μL / well, incubate at 25℃ and 450 rpm for 2 h;

[0162] (6) Washing the plate: Wash the plate with washing solution, 260 μL / well, wash the plate 5 times, leave it for 1 min each time, and then pat it dry;

[0163] (7) Add secondary antibody: Dilute the secondary antibody at a ratio of 1:2000, 100uL / well; incubate at 25℃ and 450rpm for 1h;

[0164] (8) Washing the plate: Wash the plate with washing solution, 260 μL / well, wash the plate 3 times, leave it for 1 min each time, and pat it dry;

[0165] (9) Add TMB colorimetric solution: 100 μL / well; incubate at room temperature in the dark for 15 min;

[0166] (10) Stop the reaction: Add 2M H2SO4, 100μL / well;

[0167] (11) Reading plate: 450nm, read the value.

[0168] Example 1. Sequence optimization and protein expression results of IFN-γ protein

[0169] 1. The amino acid sequence of mouse IFN-γ protein is shown below:

[0170] SEQ ID NO1:

[0171] HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIIS FYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQV QRQAFNELIRVVHQLLPESSLRKRKRSRC;

[0172] The original nucleotide sequence of mouse IFN-γ protein is shown below:

[0173] SEQ ID NO2:

[0174] cacggcacagtcattgaaagcctagaaagtctgaataactattttaactcaagtggcatagatgtggaagaaaagagtctcttcttggatatctggaggaactggcaaaaggatggtgacatgaaaatcctgcagagccagattatctctttctacctcagactctttgaagtcttgaaagacaatcaggccatcagcaa caacataagcgtcattgaatcacacctgattactaccttcttcagcaacagcaaggcgaaaaaggatgcattcatgagtattgccaagtttgaggtcaac aacccacaggtccagcgccaagcattcaatgagctcatccgagtggtccaccagctgttgccggaatccagcctcaggaagcggaaaaggagtcgctgc;

[0175] The primary mRNA secondary structure of mouse IFN-γ protein is as follows: Figure 1 As shown, its minimum free energy for secondary structure is -71.90 kcal / mol.

[0176] 2. First optimization of nucleotide sequence

[0177] The optimized mouse IFN-γ protein nucleotide sequence is shown below:

[0178] SEQ ID NO3:

[0179] cacggtaccgttattgaatccctggagagcctgaacaactacttcaactcttctggtattgatgtcgaggaaaaatctctgttcctggacatttggcgta actggcagaaagacggcgatatgaagattctgcagtcccagattattagcttctacctgcgtctgttcgaagttctgaaagacaatcaggccatctctaa caacatctccgtcatcgaatcccacctgatcactacgtttttcagcaatagcaaagcaaaaaaagacgttcatgtccatcgcgaaattcgaagtcaac aaccctcaggtgcagcgtcaggcattcaacgaactgatccgtgtcgttcaccaactgctgccggaatctagcctgcgtaaacgtaaacgctcccgttgt;

[0180] Homology comparison between the first optimized nucleic acid sequence and the original unoptimized sequence, for example Figure 2 As shown, its homology is 74.70%.

[0181] Protein expression results: The first optimized nucleotide sequence showed almost no expression of IFN-γ protein in E. coli. Therefore, the secondary structure was further optimized based on the original nucleotide sequence.

[0182] 3. Second optimization of nucleotide sequence

[0183] The second optimized mouse IFN-γ protein nucleotide sequence is shown below:

[0184] SEQ ID NO4:

[0185] cacggaacagtaatagagtcactagaaagtttgaataactatttcaactcctcaggtattgatgttgaagagaaaagcttgttcctggatatctggcgta attggcaaaaagacggcgatatgaaaatcctgcaaagccaaatcatctctttttacctccgtctgtttgaggtcctgaaggacaaccaggcgatttctaa caacatttccgtgatcgagagccacctgattaccacgtttttcagcaattcgaaagctaagaaggacgccttcatgagcattgcaaagttcgaggttaat aacccgcaggttcagcgtcaggcgtttaacgaactgatccgcgtggtgcatcagctgttgccggaatccagccttcgcaagcgcaaacgtagccgttgc;

[0186] The secondary structure of the mouse IFN-γ protein mRNA after the second optimization is as follows: Figure 3 As shown, its minimum free energy is -92.60 kcal / mol.

[0187] The homology comparison between the second optimized nucleic acid sequence and the original nucleic acid sequence is as follows: Figure 4 As shown, its homology is 77.19%.

[0188] 4. Expression results and activity assay of mouse IFN-γ protein after the second optimization.

[0189] (1) Results of recombinant expression of mouse IFN-γ protein

[0190] like Figure 5 As shown, the expression level of the original nucleic acid sequence of mouse IFN-γ protein in E. coli in vitro was 59 mg / L. According to the grayscale calculation of SDS PAGE gel image, the target protein expression accounted for only 11% of the total bacterial protein, and it was mostly present in the form of inclusion bodies.

[0191] like Figure 6 As shown, the second optimized nucleic acid sequence of mouse IFN-γ protein was expressed at 75 mg / L in E. coli in vitro. According to the grayscale calculation of the SDS-PAGE gel image, the target protein expression accounted for 35% of the total bacterial protein, and it was mainly expressed in soluble form.

[0192] Furthermore, after the above SDS-PAGE purification, the eluent still contained a small amount of other proteins besides the target protein. To remove these small amounts of other proteins, further purification using molecular sieves was performed to improve the purity of the target protein. Based on the peak results of the molecular sieves ( Figure 7 Starting from tube A09, the elution peak was collected, ultimately yielding the target protein IFN-γ with higher purity. Figure 8 ), and then perform subsequent protein activity testing.

[0193] (2) Results of protein activity of mouse IFN-γ protein

[0194] like Figure 9 As shown, the activity of mouse IFN-γ protein recombinantly expressed in Escherichia coli after the second optimization of the nucleic acid sequence was ED50 = 82.44 ng / mL.

[0195] like Figure 10 As shown, the activity of mouse IFN-γ protein recombinantly expressed in Escherichia coli with the unoptimized nucleic acid sequence was ED50 = 108.2 ng / mL.

[0196] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An optimized polynucleotide encoding a soluble IFN-γ recombinant protein, characterized in that, The nucleotide sequence of the polynucleotide is as shown in SEQ ID NO.4, or has at least 90% sequence identity with SEQ ID NO.

4.

2. The polynucleotide as described in claim 1, characterized in that, The polynucleotide encodes the IFN-γ protein shown in SEQ ID NO.

1.

3. The polynucleotide as described in claim 1, characterized in that, The polynucleotide encodes a mouse-derived IFN-γ protein.

4. The polynucleotide as described in claim 1, characterized in that, Compared to the original IFN-γ nucleic acid sequence, the polynucleotide has optimized the codon and mRNA secondary structure and eliminated the hidden splicing sites in the original nucleic acid sequence.

5. The polynucleotide as described in claim 1, characterized in that, The free energy of the secondary structure of the mRNA encoded by the polynucleotide is -92.60 kcal / mol.

6. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 1.

7. A host cell, characterized in that, The host cell contains the vector of claim 2, or has the polynucleotide of claim 1 integrated into its genome.

8. A method for preparing soluble IFN-γ protein, characterized in that, The method includes the following steps: (a) The host cells of claim 3 are cultured under suitable culture conditions to obtain a culture; and (b) Soluble IFN-γ protein was isolated and purified from the culture.

9. A recombinant IFN-γ protein, characterized in that, The recombinant protein is produced by the cells described in claim 7, or prepared by the method described in claim 8.

10. The use of the polynucleotide of claim 1, the expression vector of claim 6, the host cell of claim 7, or the recombinant protein of claim 9, characterized in that, Used in the preparation of formulations or compositions.

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