CN111690069B - A kind of IL-15/SuIL-15Rα-mFc-γ4 complex protein and its construction method and application - Google Patents

Abstract

The present invention discloses an IL-15/SuIL-15Rα-mFc-γ4 complex protein, comprising the amino acid sequence shown in SEQ ID No. 1 or an amino acid sequence with more than 90% identity with SEQ ID No. 1. The invention also discloses the construction method of the above IL-15/SuIL-15Rα-mFc-γ4 complex protein. The invention also discloses two expression vectors. The invention also discloses a yeast strain. The invention also discloses the application of the above IL-15/SuIL-15Rα-mFc-γ4 complex protein in the preparation of antiviral and antitumor drugs. The invention also discloses the application of the above expression vector or yeast strain in expressing IL-15/SuIL-15Rα-mFc-γ4 complex protein. The complex protein significantly prolonged the half-life of IL-15.

Description

A kind of IL-15/SuIL-15Rα-mFc-γ4 complex protein and its construction method and application

technical field

The present invention relates to the technical field of protein engineering, in particular to an IL-15/SuIL-15Rα-mFc-γ4 complex protein and a construction method and application thereof.

Background technique

Interleukin 15 (IL-15) is an important immunologically active protein, which has been clinically used for antiviral and antitumor treatment research. However, the low potency and short half-life of monomeric IL-15 molecules severely limit its application as a clinical drug. In order to improve the therapeutic effect of IL-15, researchers have successively developed a variety of IL-15 complexes. For example, as early as 2006, researchers such as Erwan Mortier linked IL-15 to the sushi domain of IL-15 receptor α to form complex RLI, which significantly increased the activity of IL-15.

In addition to forming complexes with receptors, fusion with the Fc region of antibodies is an important method to prolong the half-life of protein drugs. There have been several successful cases in this regard. For example, Enbrel (TNFR-Fc), the world's best-selling Fc fusion protein, and the fusion drug protein of recombinant blood factor VIII and Fc developed by Biogen Idea recently approved for marketing by the FDA. There have also been some attempts to increase the half-life of IL-15 by fusion to Fc, for example in 2008 Sigrid Dubois et al developed a complex consisting of IL-15 and dimerized IL-15Rα-IgG1-Fc, 2011 Altor BioScience developed ALT-803 (now renamed N-803). Both effectively increased the in vivo half-life of IL-15.

Fc fusion proteins extend plasma half-life in vivo through the neonatal Fc receptor (FcRn). The specific mechanism is that the Fc fusion protein binds to the FcRn of phagocytic cells in vivo and is finally released into the extracellular cycle to avoid being degraded by lysosomes.

SUMMARY OF THE INVENTION

Based on the technical problems existing in the background technology, the present invention proposes an IL-15/SuIL-15Rα-mFc-γ4 complex protein and its construction method and application. The present invention constructs an IL-15 based on Pichia pastoris. /SuIL-15Rα-mFc-γ4 complex protein, significantly prolongs the half-life of IL-15.

An IL-15/SuIL-15Rα-mFc-γ4 complex protein proposed by the present invention comprises the amino acid sequence shown in SEQ ID No. 1 or an amino acid sequence with more than 90% identity with SEQ ID No. 1.

The above-mentioned amino acid sequence shown in SEQ ID No. 1 has a nucleotide sequence shown in SEQ ID No. 2.

The present invention also proposes a method for constructing the above-mentioned IL-15/SuIL-15Rα-mFc-γ4 complex protein, including the following steps:

S1, mutate human IL-15 to obtain an IL-15 variant whose nucleotide sequence is shown in SEQ ID No.3;

S2, mutate the Fc fragment of human IgG4 to obtain an Fc variant whose nucleotide sequence is shown in SEQ ID No.5;

S3. SuIL-15Rα-mFc-γ4 is obtained by linking the Fc variant to the sushi domain of IL-15Rα through a linker peptide, wherein the nucleotide sequence of the linker peptide is shown in SEQ ID No. 7, and SuIL-15Rα-mFc -The nucleotide sequence of γ4 is shown in SEQID No.9;

S4. Co-express the IL-15 variant and SuIL-15Rα-mFc-γ4 to obtain the IL-15/SuIL-15Rα-mFc-γ4 complex protein.

The amino acid sequence of the above-mentioned IL-15 variant is shown in SEQ ID No. 4, the amino acid sequence of the Fc variant is shown in SEQ ID No. 6, the amino acid sequence of the connecting peptide is shown in SEQ ID No. 8, and SuIL-15Rα- The amino acid sequence of mFc-γ4 is shown in SEQ ID No.10.

The above-mentioned mutation of the Fc fragment of human IL-15 and human IgG4 is performed according to the conventional mutation method in the art.

The structure of the above-mentioned IL-15/SuIL-15Rα-mFc-γ4 complex protein and the schematic diagram of its construction method are shown in Figure 1. Figure 1 is a schematic diagram of the construction of an expression vector for the IL-15/SuIL-15Rα-mFc-γ4 complex protein. and a schematic diagram of the structure, wherein, A is a schematic diagram of the construction of an expression vector; B is a schematic diagram of the structure. In Figure 1A, the expression vector pPIC9-SuIL-15Rα-mFc-γ4 and the expression vector pPICZα-IL-15 were respectively inserted into the Pichia GS115 strain by homologous recombination using the HIS4 sequence and 5'AOX1 sequence on their vectors, respectively. The corresponding site in the genome was constructed to construct a Pichia strain capable of expressing IL-15/SuIL-15Rα-mFc-γ4 complex protein.

The present invention also proposes an expression vector, which includes a 5'AOX1 promoter, a transcription terminator, an antibiotic resistance gene and a secretion signal peptide, and also includes the construction method of the above-mentioned IL-15/SuIL-15Rα-mFc-γ4 complex protein Nucleotide sequences of IL-15 variants in .

The present invention also proposes an expression vector, which includes a 5'AOX1 promoter, a transcription terminator, an antibiotic resistance gene and a secretion signal peptide, and also includes the structure of the above-mentioned IL-15/SuIL-15Rα-mFc-γ4 complex protein Nucleotide sequence of SuIL-15Rα-mFc-γ4 in Methods.

The present invention also provides a yeast strain comprising the above two expression vectors.

Preferably, the yeast strain is a Pichia strain.

The present invention also proposes the application of the above IL-15/SuIL-15Rα-mFc-γ4 complex protein in the preparation of antiviral and antitumor drugs.

The present invention also proposes the application of the above expression vector or the above yeast strain in expressing IL-15/SuIL-15Rα-mFc-γ4 complex protein.

After a long-term accumulation of fusion protein research, the inventors constructed an IL-15/SuIL-15Rα-mFc-γ4 complex protein based on Pichia pastoris, which significantly prolonged the half-life of IL-15; 15 Mutation, the 72nd asparagine on the IL-15 amino acid sequence was mutated to aspartic acid to improve the biological activity of IL-15, the 71st, 79th, and 112th asparagine Both were mutated to glutamine to reduce the potential immunogenicity of glycosylation modifications to obtain IL-15 variants; the Fc fragment of human IgG4 was mutated, and the 235th leucine on the amino acid sequence of the Fc fragment was mutated. Mutation of acid to glutamic acid and mutation of asparagine at position 297 to glutamine to reduce effects such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to obtain Fc variants ; By selecting a suitable linking peptide, the Fc variant is linked to the sushi domain of IL-15Rα to obtain SuIL-15Rα-mFc-γ4, wherein mFc is a single-chain Fc, and a flexible GS linking peptide can be selected to maintain IL-15/ The activity of SuIL-15Rα-mFc-γ4 complex protein; and the molecular weight of single-chain Fc is smaller, it may be easier to enter tumor tissue or infected tissue in vivo and play a better therapeutic effect; the present invention adopts simple and cheap The Pichia pastoris expression system produced the IL-15/SuIL-15Rα-mFc-γ4 complex protein based on antibody single-chain Fc; established a Chinese-style production process and conducted pharmacodynamics and pharmacokinetics studies. It lays the foundation for its future clinical trials and applications.

Description of drawings

Figure 1 is a schematic diagram of construction of an expression vector and a schematic diagram of the structure of the IL-15/SuIL-15Rα-mFc-γ4 complex protein, wherein A is a schematic diagram of the construction of an expression vector; B is a schematic diagram of the structure.

Figure 2 shows the results of screening and identification of Pichia strains expressing IL-15/SuIL-15Rα-mFc-γ4 complex protein, wherein A is the screening result of Dot-Blot, and B is the identification result of Western Blot.

Figure 3 shows the results of the time point detection of IL-15/SuIL-15Rα-mFc-γ4 complex protein accumulation during fermentation.

Figure 4 shows the SDS-PAGE and Western Blot identification results of the IL-15/SuIL-15Rα-mFc-γ4 complex protein, wherein A is the SDS-PAGE identification, and B is the Western Blot identification.

Figure 5 shows the results of the in vitro biological activity assay of IL-15/SuIL-15Rα-mFc-γ4 complex protein.

Figure 6 shows the results of the detection of the circulating half-life of IL-15/SuIL-15Rα-mFc-γ4 complex protein in mice in vivo.

Figure 7 shows the in vivo biological activity detection results of IL-15/SuIL-15Rα-mFc-γ4 complex protein, wherein A is the photo of mouse spleen and spleen weight, and B is the proportion and number of immune cell subsets in mouse spleen.

Detailed ways

Hereinafter, the technical solutions of the present invention will be described in detail through specific embodiments.

The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

All primer synthesis and sequencing work in the following examples were completed by Shanghai Shenggong; the used Pichia-related vectors and strains were derived from Invitrogen (Life technologies); the formula of the medium used was referred to PichiaExpression Kit, A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris, Invitrogen).

Experimental animals: The experimental C57BL/6 mice were bred and housed in the SPF animal room of the School of Life Sciences, University of Science and Technology of China.

Experimental reagents: DNA gel recovery kit (Axygen company), PCR cleaning and recovery kit (Axygen company), plasmid mini-extraction kit (Axygen company), plasmid mid-volume extraction kit (Axygen company), anti-human IgG4 Fc antibody (Abcam company), anti-human IL-15 monoclonal antibody (Boster Bioengineering Co., Ltd.), anti-rabbit secondary antibody (Biolegend company), RPMI 1640 medium (HyClone company), fetal bovine serum (BI company) , IL-2 (Jiangsu Kingsley Pharmaceutical Co., Ltd.), rhIL-15 (PeproTech Company), IL-15 capture antibody and IL-15 detection antibody (R&D Systems Company).

Main equipment: UV spectrophotometer (SHIMADZU, Japan), microplate reader (Bio Tek, USA), automatic tissue disruptor (Miltenyi Biotec, Germany), flow cytometer (Beckman Coulter, USA), cell counting instrument (Countstar, China).

Example 1 Construction of SuIL-15Rα-mFc-γ4 fusion protein expression vector

1 Amplification of SuIL-15Rα-mFc-γ4 fusion gene

1.1 Using pPIC9-SuIL-15R as a template, use primer 1 and primer 2 to amplify fragment 1. The PCR program is: 94°C for 5 min, (94°C for 30s, 55°C for 40s, 72°C for 20s) This program is set for 32 cycles , 72℃10min;

1.2 Using pPICZα-Fc-mut as a template, use primer 3 and primer 4 to amplify fragment 2. The PCR program is: 94°C for 5 min, (94°C for 30s, 55°C for 40s, 72°C for 50s) This program is set for 32 cycles , 72℃10min;

1.3 Perform agarose gel electrophoresis on the amplified products in 1.1 and 1.2. After electrophoresis, use a DNA gel recovery kit (operate according to the kit instructions) to recover two gene fragments, namely fragment 1 and fragment 2;

1.4 Mix Fragment 1 and Fragment 2 as a template, and perform amplification without adding primers. The PCR program is set to: 94°C for 5 min, (94°C for 30s, 55°C for 40s, 72°C for 60s) This program is set for 8 cycles, 72°C for 10min; then add primer 1 and primer 4 to amplify, the PCR program is set to: 94°C for 5min, (94°C for 30s, 55°C for 40s, 72°C for 60s) this program is set for 32 cycles, 72°C for 10min; The amplified product was subjected to agarose gel electrophoresis, and then a DNA gel recovery kit was used to recover a gene with XhoI and NotI restriction sites at both ends, named SuIL-15Rα-mFc-γ4;

The sequences of the above primers 1-4 are shown in the following table:

The above-mentioned "pPIC9-SuIL-15R" is the plasmid pPIC9 containing the SuIL-15R gene, and SuIL-15R is the sushi domain gene of IL-15Rα;

The above-mentioned "pPICZα-Fc-mut" is the plasmid pPICZα containing the IgG4Fc gene mutated at sites 228, 235 and 297;

The role of pPIC9-SuIL-15R, pPICZα-Fc-mut is to link the Fc variant to the sushi domain of IL-15Rα through a linker peptide, wherein the 228 position mutation is not linked to SuIL-15Rα-mFc- in the sequence of γ4;

Both pPIC9-SuIL-15R and pPICZα-Fc-mut were obtained by the inventors constructing genes into corresponding plasmids according to conventional expression vector construction methods.

2 Construction of Pichia pastoris expression vector for SuIL-15Rα-mFc-γ4 fusion protein

2.1 The gene SuIL-15Rα-mFc-γ4 obtained in step 1 and the purchased plasmid pPIC9 vector were double-enzyme digested with restriction enzymes Xho I and Not I; then the digested gene was recovered using a PCR cleaning recovery kit SuIL-15Rα-mFc-γ4, use DNA gel recovery kit to recover the digested plasmid pPIC9 vector;

2.2 The digested gene SuIL-15Rα-mFc-γ4 was ligated with the digested plasmid pPIC9 vector using T4 DNA ligase to obtain the ligated product; 10 μl of the ligated product was added to 100 μl of E. coli DH5α competent cells , stand on ice for 30min, flick and mix every 10min, then heat shock at 42°C for 90s in a water bath, immediately place it on ice for 3-5min after taking it out, add 400μl sterile LB liquid medium ( No resistance), incubate at 37 °C for 45 min on a shaker, take 100 μl of bacterial suspension and spread it on a LA medium plate (LB plate with ampicillin resistance), and place it in a 37 °C incubator for cultivation; after the plate grows clonal colonies , select a single colony and clone it into a shake tube containing LA liquid medium (LB medium with ampicillin resistance), cultivate at 37°C for 6-8h, and then use the bacterial liquid as a template for PCR identification, and the obtained positive clone is named pPIC9- SuIL-15Rα-mFc-γ4, the bacterial liquid corresponding to the positive clone was extracted with a plasmid mini-extraction kit, and sent to Shanghai Sangon Biotechnology for sequencing.

Experimental results: The sequencing results were consistent with the theoretical expected results, and the clones were constructed correctly.

Example 2 Expression and purification of IL-15/SuIL-15Rα-mFc-γ4 complex protein

1 complex protein expression

1.1 Gene expression and linearization: the positive clones pPIC9-SuIL-15Rα-mFc-γ4 and plasmid pPICZα-IL-15 (which are the plasmid pPICZα containing the IL-15 variant gene) screened in Example 1 can be obtained by conventional methods. It is constructed, and its function is to use the 5'AOX1 sequence to integrate the IL-15 variant gene into the corresponding site in the Pichia pastoris cells, thereby expressing the IL-15 protein in the Pichia pastoris cells. Construction method, self-constructed) was added to 50 mL of LA medium to expand the culture, and the plasmid was extracted using a plasmid medium extraction kit to obtain the expression plasmid pPIC9-SuIL-15Rα-mFc-γ4 and expression plasmid pPICZα-IL-15; Plasmid pPIC9-SuIL-15Rα-mFc-γ4 and expression plasmid pPICZα-IL-15 were linearized with endonuclease SalI and SacI, respectively, and recovered by ethanol precipitation; the specific operation steps of recovery are as follows: add 1/10 volume of 3M Sodium acetate (pH 5.2) was mixed thoroughly in the enzyme digestion reaction system, then 2-2.5 times the volume of ice ethanol was added, and after mixing, it was placed in a -20°C refrigerator for 1-2 hours; Discard the supernatant, add 1 mL of 70% ethanol to resuspend the pellet, centrifuge at 12,000g for 10 min at 4°C, discard the supernatant, and repeat the washing; open the lid of the EP tube at room temperature to evaporate the residual ethanol, add an appropriate amount of deionized water to dissolve, and Use Nanodrop to measure the concentration, and adjust the concentration between 0.5-1.0 μg/μL to obtain the linearized plasmid pPIC9-SuIL-15Rα-mFc-γ4 and the linearized plasmid pPICZα-IL-15;

1.2 Preparation of yeast competence: Take out the frozen GS115 Pichia strain from the refrigerator, streak it on a YPD plate, and place it in a 30°C constant temperature incubator for cultivation; observe the yeast clones growing to a diameter of about 1-2 mm, pick them out The single clone was placed in 3-4 mL of YPD medium, and placed in a yeast shaker for constant temperature cultivation at 30 °C. After 24-48 hours, 1 mL of bacterial liquid was inoculated into 50 mL of YPD medium, and continued to be incubated at a constant temperature of 30 °C in a yeast shaker to monitor the bacteria. When the OD ₆₀₀ value reaches about 1-1.5, transfer the bacterial liquid to a 50mL centrifuge tube, centrifuge at 1500g for 5min at 4°C, discard the supernatant after centrifugation, add 40mL ice water to resuspend, and centrifuge at 1500g for 5min at 4°C. Discard the supernatant and repeat washing with ice water; resuspend the pellet after centrifugation with 20 mL of 1M cold sorbitol, centrifuge at 1500g for 5 min at 4°C, discard the supernatant, and finally add 500 μL of 1M cold sorbitol to resuspend the cells to obtain yeast competent cells ;

1.3 Electroporation of pPIC9-SuIL-15Rα-mFc-γ4: Mix 10 μL of the linearized plasmid pPIC9-SuIL-15Rα-mFc-γ4 (5-10 μg) with 100 μl of yeast competent cells, and add it to a sterile pre-cooled electroporation Set the parameters of the electroporator as: 2000V, 200Ω, 25μF; start electroporation, immediately add 1 mL of 1M cold sorbitol to the electroporation cup, mix gently, take the bacterial solution and spread it on the MD plate. Incubate in a 30°C constant temperature yeast incubator until colonies appear; pick multiple clones into 4mL MGY medium, incubate at 30°C for 18-24h on a yeast shaker, take the bacterial solution, centrifuge at 12000g for 1min, take the supernatant with anti-human The antibody of IgG4 Fc was detected by Western Blot, and the positive clones obtained were made into yeast competent cells containing pPIC9-SuIL-15Rα-mFc-γ4 according to the yeast competent preparation method in 1.2; then 10 μl were linearized according to the above electroporation method. The plasmid pPICZα-IL-15 was introduced into yeast competent cells containing pPIC9-SuIL-15Rα-mFc-γ4, and the electroporated bacterial solution was spread on a YPD plate (containing zeocin);

1.4 Screening of IL-15/SuIL-15Rα-mFc-γ4 positive expression clones: Place the YPD plate (containing zeocin) coated with bacterial liquid in 1.3 in a constant temperature yeast incubator at 30°C for cultivation until colonies appear, and pick more The clones were placed in 4 mL of YPD medium, and incubated in a yeast shaker at a constant temperature of 30°C for 24-48 hours, 3 mL of bacterial solution was drawn from it, centrifuged at 1500 g for 5 min, the supernatant was discarded, and 4 mL of BMMY medium was added to resuspend the pellet, and placed in a shaker to continue Culture, add methanol solution every 24h (the final concentration of methanol is 1%), after 48h, centrifuge the bacterial solution at 12000g for 1min, draw the supernatant and prepare samples, carry out Dot-Blot primary screening, and select those with relatively high expression levels. Yeast strains, and then spread the yeast strains with relatively high expression on the YPD plate containing zeocin, after the clones grow, select multiple clones from them and shake the bacteria according to the above method and obtain the supernatant of the bacterial liquid for Western Blot identification. , respectively incubated with anti-IgG4 Fc antibody and anti-human IL-15 antibody to screen out high-expressing strains, and cryopreserved the remaining 1 mL of the corresponding strain; A Pichia strain of 15Rα-dFc-γ4 complex protein;

The above-mentioned Dot-Blot and Western Blot identification results are shown in Figure 2. Figure 2 shows the screening and identification results of Pichia strains that can express the IL-15/SuIL-15Rα-mFc-γ4 complex protein, where A is Dot-Blot. Screening results, B is the Western Blot identification results.

It can be seen from Figure 2 that the Dot-Blot preliminary screening results show that the Pichia strains numbered 24 to 68 express the IL-15/SuIL-15Rα-mFc-γ4 complex protein to varying degrees, and select the ones with relatively high expression levels. The strains numbered 44 and 62 were further screened and identified by Western Blot. Finally, the Pichia strain numbered 62-4 was selected as the Pichia pastoris that can highly express the IL-15/SuIL-15Rα-mFc-γ4 complex protein strains.

2Fermentation, purification and identification of complex proteins

2.1 Fermentation of complex protein: Ferment the high-expressing strains screened in step 1, and the fermentation operation steps are carried out according to the fermentation guidelines of Invitrogen; inoculate the expression strains in 4 mL of MGY medium, and cultivate at a constant temperature of 30 °C in a yeast shaker. 24-48h, the bacterial liquid obtained in this step is the fermentation first-class seed solution; take 1mL of the first-class seed solution and inoculate it into 400mL of BMGY medium, and cultivate at 30℃ for 12-24h in a constant temperature shaker until the OD600 is 2-6, this step The obtained bacterial liquid is the fermentation secondary seed liquid; 400 mL of the secondary seed liquid is all inoculated into the fermenter containing BMGY medium, the parameters of the fermenter are set as the culture temperature of 28 ° C, pH 6.0, and the synchronous monitoring of the fermentor in the fermenter is performed. When the dissolved oxygen in the tank rises rapidly, start to add glycerol, monitor the wet weight of the bacteria, and start to induce the induction by adding methanol according to the three-step method recommended by Invitrogen. The pH value of the culture supernatant is 7-8, centrifuged at 10,000g for 20min, the supernatant was collected, and filtered with 0.45μm and 500KDa microfilters successively;

2.2 Purification: Load the supernatant filtered in step 2.1 onto the affinity chromatography column Hitrap MabSelect. The specific steps are: use 5 column volumes of deionized water to rinse the column, and then use 3 column volumes of PBS buffer to rinse After passing through the column, use 3 column volumes of PBS buffer to wash the column, and use the eluent to elute the target protein. The eluent formula is: 100mM sodium citrate/lemon acid, pH=3.0, after the elution is completed, add 1M Tris-HCl to the collected eluent to adjust the pH to neutrality to obtain a neutral eluent; then finely purify, the specific steps are: using AKTAPURE 25, The Superdex200 molecular sieve was equilibrated with PBS buffer. After equilibration, the neutral eluate was applied to the sample, washed with PBS buffer, and the eluted sample was collected to obtain IL-15/SuIL-15Rα-mFc-γ4 complex protein.

2.3 Test: monitor the accumulation of complex protein in the fermentation process of step 2.1 complex protein during methanol induction. The results are shown in Figure 3. Figure 3 shows the accumulation of IL-15/SuIL-15Rα-mFc-γ4 complex protein during the fermentation process Time point detection results; it can be seen from Figure 3 that the accumulation of IL-15/SuIL-15Rα-mFc-γ4 complex protein continued to increase during the induction period of 0-32h; The accumulation of 15Rα-mFc-γ4 complex protein no longer increased, even decreased.

2.4 Identification: SDS-PAGE and Western Blot identification of the complex protein obtained in step 2.2, the results are shown in Figure 4; Figure 4 is the SDS-PAGE and Western Blot of the IL-15/SuIL-15Rα-mFc-γ4 complex protein Identification results, among which, A is identified by SDS-PAGE, and B is identified by Western Blot; it can be seen from Figure 4A that the protein purity of IL-15/SuIL-15Rα-mFc-γ4 complex can reach more than 90%, and it is mainly monomeric. The form exists, which is consistent with expectations; it can be seen from Figure 4B that the complex protein contains IL-15 components and Fc-γ4 components, and the resulting recombinant protein should be IL-15/SuIL-15Rα-mFc-γ4 complex protein.

Example 3 In vitro biological activity detection of IL-15/SuIL-15Rα-mFc-γ4 complex protein

1 Experimental method

The cell proliferation-promoting activity of IL-15/SuIL-15Rα-mFc-γ4 complex protein was determined by CTLL-2 cells

1.1 Recovery and passage of CTLL-2 cells: Take out CTLL-2 cells from the liquid nitrogen tank (a gift from the Institute of Immunology, School of Life Sciences, University of Science and Technology of China), shake them back and forth in a 37°C water bath to quickly melt them, and add them to the 5mL complete medium (complete medium formula: RPMI 1640 medium + 10% fetal bovine serum (BI company) + 200IU/mL IL-2) centrifuge tube, room temperature, 200g centrifugation for 5min, after centrifugation, use 1mL Resuspend in complete medium, count, and inoculate 4×10 ⁴ cells/mL in a 25cm ² culture flask; when the cell density reaches 2×10 ⁵ cells/mL, subculture, take a 15mL centrifuge tube, add the cell suspension to In a centrifuge tube, centrifuge at 200g for 5min at room temperature. After centrifugation, resuspend with 1mL of complete medium, count, inoculate at 1-2×10 ⁴ cells/mL, and then subculture at the above cell density every 2-3 days;

1.2 Determination of the cell proliferation-promoting activity of IL-15/SuIL-15Rα-mFc-γ4 complex protein: Take CTLL-2 cells cultured to logarithmic growth phase, centrifuge at room temperature, 200g for 5min, after centrifugation, discard the supernatant, add Cells were resuspended in 5 mL RPMI-1640 medium, centrifuged at 200g for 5 min at room temperature, and washed twice; resuspended with 1 mL basal medium (basal medium formula: RPMI-1640 medium + 10% FBS), counted, and adjusted cell density , 60 μL of cell suspension was added to a 96-well plate to make the number of cells 1.0×10 ⁴ cells/well; rhIL-15 and IL-15/SuIL-15Rα-mFc-γ4 complex proteins were added to the basal medium by 2 times Gradient dilution, adding 40 μL to each well, and incubating for 48 h. After the incubation, the MTT method was used for color development, and the absorbance at OD ₄₉₀ nm was measured using a microplate reader.

2 Experimental results:

The MTT colorimetric results were calculated by Graphpad Prism software, and the four-parameter logistic equation was used for curve fitting to calculate the EC ₅₀ value. The results are shown in Figure 5, which is the IL-15/SuIL-15Rα-mFc-γ4 complex. In vitro biological activity test results of the protein;

It can be seen from Figure 5 that the EC ₅₀ value of rhIL-15 to stimulate the proliferation of CTLL-2 cells is about 5.99 pM, and the EC ₅₀ value of IL-15/SuIL-15Rα-mFc-γ4 complex protein to stimulate the proliferation of CTLL-2 cells is about 5.99 pM. At 12.74 pM, the activity of IL-15/SuIL-15Rα-mFc-γ4 complex protein was comparable to other reported IL-15 complexes, and the activity was even better.

Example 4 In vivo half-life detection of IL-15/SuIL-15Rα-mFc-γ4 complex protein

1 Experimental method:

Select healthy C57BL/6 mice with a body weight of 18-25g for 6-8 weeks and randomly divide them into 2 groups with 4 mice in each group. According to the principle of the same molecular weight of IL-15 injected, that is, rhIL-15 0.28mg The dose of IL-15/SuIL-15Rα-mFc-γ41mg/kg was injected into the tail vein of mice, and the blood was collected at different time points. The blood collection time points of the mice in the rhIL-15 group were 0.25, 0.5, 1, 2, 4h, the time points of blood collection of mice in IL-15/SuIL-15Rα-mFc-γ4 group were: 0.5, 2, 4, 8, 10, 12, 24, 48h, centrifugation after blood collection Serum was collected, the protein concentration in serum samples was determined by ELISA, and the pharmacokinetic data were processed according to the non-compartmental model using PKsolver software.

2 Experimental results:

The half-life of commercialized rhIL-15 and IL-15/SuIL-15Rα-mFc-γ4 complex was measured using C57BL/6 mice. Circulating half-life test results of γ4 complex in mice;

It can be seen from Figure 6 that the half-life of rhIL-15 is about 0.7h, the half-life of IL-15/SuIL-15Rα-mFc-γ4 is about 9.16h, and the half-life of IL-15/SuIL-15Rα-mFc-γ4 complex protein is The half-life was significantly longer than that of commercial rhIL-15.

Example 5 In vivo biological activity detection of IL-15/SuIL-15Rα-mFc-γ4 complex protein

Healthy C57BL/6 mice weighing 18-25g for 6-8 weeks were selected and randomly divided into 3 groups with 5 mice in each group. The dose of γ4 1 mg/kg was injected into the tail vein of the mice, and the control group was injected with an equal volume of PBS buffer. After 72 hours, the eyeballs were removed to collect blood, and then the mice were killed by cervical dislocation, and the spleens of the mice were removed and placed in ice-cold PBS buffer. In , the mouse spleen was photographed and weighed, and then the spleen was further processed for flow detection. The specific processing methods were as follows:

The mouse spleen was ground with an automatic tissue disruptor. After grinding, a 100-mesh nylon mesh was used for filtration. After filtration, centrifuge at 4°C, 2200 rpm for 10 min; discard the supernatant, scatter the cells, and add 1 mL of red blood cell lysate. Mix well, lyse at room temperature in the dark for 5 min until clear, add 10 mL of PBS buffer to stop the lysis, filter through a 200-mesh nylon mesh into a 15 mL centrifuge tube, discard the supernatant after centrifugation, add 1 mL of PBS buffer to wash the cells, 4°C, 3000rpm , centrifuged for 5 min, resuspended in 1×PBS buffer containing 10% rat serum, blocked for 30 min at 4°C in the dark, took part of the cells and diluted them and counted them with a cell counter. After blocking, flow antibody-labeled CD3 , CD8, CD4, CD44, NK1.1, CD19, CD45 molecules (purchased from Biolegend) at 4°C, blocked from light for 1 h, washed with 1×PBS buffer, centrifuged and resuspended in 1×PBS buffer. On-board detection;

The test results are shown in Figure 7. Figure 7 is the in vivo biological activity test results of the IL-15/SuIL-15Rα-mFc-γ4 complex protein, wherein A is the photo of the mouse spleen and the weight of the spleen, and B is the immune cell subtype of the mouse spleen. the proportion and number of groups;

As can be seen from Figure 7A, the spleen of mice injected with IL-15/SuIL-15Rα-mFc-γ4 complex protein was significantly larger than that of the control group (PBS group) and the commercial rhIL-15 group, and the weight of the spleen was significantly increased. It can be seen from Figure 7B that in the spleen of mice injected with IL-15/SuIL-15Rα-mFc-γ4 complex protein, CD8 ⁺ T cells, CD8 ⁺ CD44 ⁺ T cells (memory phenotype T cells) ), the proportion of NK cells and NKT cells and the number of cells were significantly increased compared with the control group (PBS group) and commercial rhIL-15 group; the above results showed that IL-15/SuIL-15Rα-mFc-γ4 complex protein The in vivo biological activity of IL-15 was significantly better than that of monomeric IL-15 molecules.

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. The equivalent replacement or change of the inventive concept thereof shall be included within the protection scope of the present invention.

sequence listing

<110> University of Science and Technology of China

<120> A kind of IL-15/SuIL-15Rα-mFc-γ4 complex protein and its construction method and application

<130> 2019

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Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

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Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

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Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

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Asn Leu Ile Ile Leu Ala Gln Asp Ser Leu Ser Ser Asn Gly Gln Val

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Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

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Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Gln

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Thr Ser Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile

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Trp Val Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn

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Ser Gly Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val

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Leu Asn Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys

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Cys Ile Arg Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

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Gly Gly Ser Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

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Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

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Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

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Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

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Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr

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Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

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Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

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Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

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Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

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Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

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Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

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Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

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Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

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gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctaccccagc 1020

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Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

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Asn Leu Ile Ile Leu Ala Gln Asp Ser Leu Ser Ser Asn Gly Gln Val

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cccgaggtcc agttcaactg gtacgtggat ggcgtggagg tgcataatgc caagacaaag 180

ccgcgggagg agcagttcca aagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240

caggactggc tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg cctcccgtcc 300

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ggcttctacc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 480

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcagacta 540

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Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

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Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

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Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

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Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met

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Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

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Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

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Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

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Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val

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Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

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Lys Ser Leu Ser Leu Ser Leu Gly Lys

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

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gaccccgagg tccagttcaa ctggtacgtg gatggcgtgg aggtgcataa tgccaagaca 420

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Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

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Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

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Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

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Arg Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

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Ser Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

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Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

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Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp

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Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

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Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

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His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

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Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

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Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu

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Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

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Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

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Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

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Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn

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Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

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Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

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Claims (8)

1. An IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein is characterized in that the amino acid sequence is shown as SEQ ID No. 1.

2. A method of constructing the IL-15/sull-15 ra-mFc- γ 4 complex protein of claim 1, comprising the steps of:

s1, mutating the human IL-15 to obtain an IL-15 variant with a nucleotide sequence shown as SEQ ID No. 3;

s2, mutating an Fc fragment of human IgG4 to obtain an Fc variant with a nucleotide sequence shown as SEQ ID No. 5;

s3, connecting the Fc variant with a sushi structural domain of IL-15 Ra through a connecting peptide to obtain SuIL-15 Ra-mFc-gamma 4, wherein the nucleotide sequence of the connecting peptide is shown as SEQ ID No.7, and the nucleotide sequence of the SuIL-15 Ra-mFc-gamma 4 is shown as SEQ ID No. 9;

s4, co-expressing the IL-15 variant and SuIL-15R alpha-mFc-gamma 4 to obtain the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein.

3. An expression vector comprising a 5' AOX1 promoter, a transcription terminator, an antibiotic resistance gene, and a secretion signal peptide, further comprising the nucleotide sequence of the IL-15 variant of the method of constructing the IL-15/sull-15 ra-mFc- γ 4 complex protein of claim 2.

4. An expression vector comprising a 5' AOX1 promoter, a transcription terminator, an antibiotic resistance gene, and a secretion signal peptide, further comprising the nucleotide sequence of sull-15 ra-mFc-gamma 4 in the method for constructing the IL-15/sull-15 ra-mFc-gamma 4 complex protein according to claim 2.

5. A yeast strain comprising the expression vector of claim 3 and the expression vector of claim 4.

6. The yeast strain of claim 5, wherein the yeast strain is a Pichia strain.

7. An application of the IL-15/SuIL-15 Ra-mFc-gamma 4 complex protein as defined in claim 1 in preparing antiviral and antitumor medicines.

8. Use of an expression vector according to claim 3 or 4 or a yeast strain according to claim 5 or 6 for the expression of the IL-15/sull-15 ra-mFc-gamma 4 complex protein.

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