CN111690068B - A kind of IL-15/SuIL-15Rα-dFc-γ4 complex protein and its construction method and application - Google Patents

Abstract

The present invention discloses an IL-15/SuIL-15Rα-dFc-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-dFc-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-dFc-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-dFc-γ4 complex protein. The complex protein significantly prolongs the half-life of IL-15.

Description

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

technical field

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

Background technique

Interleukin 15 (IL-15) is a pleiotropic cytokine involved in the regulation of various immune functions, especially in promoting the development, proliferation and activation of NK cells, NKT cells and memory CD8 ⁺ T cells have an important role. As a member of the universal receptor gamma chain cytokine family (this family includes IL-2, IL-4, IL-7, IL-9, IL-15, IL-21), IL-15 is structurally similar to IL-2, and It shares the receptor β subunit (IL-2/IL-15Rβ) with IL-2, so it has many similar biological functions with IL-2. Different from the cis-presenting mechanism of IL-2, IL-15 has a unique trans-presenting mechanism, which can further bind to IL-15 on the surface of effector cells by binding to its high-affinity receptor α subunit (IL-15Rα). The 2Rβ subunit and the universal γ chain form a complex to transmit signals into the cell. Although IL-2 and IL-15 have similar roles in stimulating immune cell proliferation and activation, there are still significant differences between the two in the adaptive immune response, and IL-2 can also induce cell death through activation ( AICD) mechanism and maintain the stability of Treg cells to limit the immune response, while IL-15 has no obvious effect on Treg cells, but also inhibits IL-2-induced AICD and promotes NK cells, CD8 ⁺ CD44 ^hi memory T cells This suggests that IL-15 may be superior to IL-2 in anti-tumor and anti-viral therapy. The clinical trial results of recombinant human IL-15 (rhIL-15) showed that after injection of rhIL-15, γδ T cells, CD8+ T cells and NK cells in the blood of patients were significantly increased. It is short and has certain toxic reactions when injected at high doses, which limits its clinical application.

In order to overcome the above shortcomings, researchers have successively developed a variety of IL-15 complexes based on the physiological properties of IL-15. For example, in 2006, Erwan Mortier et al. The complex RLI, developed in 2008 by Sigrid Dubois et al. A complex composed of IL-15 and dimerized IL-15Rα-IgG1-Fc (IL-15/IL-15Rα-IgG1-Fc), 2011 Researchers at AltorBioScience have developed a complex composed of IL-15N72D (a mutant form of IL-15) molecule and dimerized IL-15Rα (sushi domain)-IgG1-Fc (now renamed ALT-803) N-803). The above-mentioned types of IL-15 complexes all show better biological activity and prolonged half-life than pure IL-15 molecules, and many studies have entered clinical trials and achieved encouraging results, which shows that IL-15 has great development potential in anti-tumor and anti-viral therapy.

SUMMARY OF THE INVENTION

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

An IL-15/SuIL-15Rα-dFc-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-dFc-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 hinge region and Fc fragment of human IgG4 to obtain an IgG4 Fc variant whose nucleotide sequence is shown in SEQ ID No.5;

S3. Connect the IgG4Fc variant and the sushi domain of IL-15Rα through the hinge region in the IgG4Fc variant to obtain SuIL-15Rα-dFc-γ4, wherein the nucleotide sequence of SuIL-15Rα-dFc-γ4 is shown in SEQ ID No. .7 shown;

S4. Co-express the IL-15 variant and SuIL-15Rα-dFc-γ4 to obtain the IL-15/SuIL-15Rα-dFc-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 IgG4Fc variant is shown in SEQ ID No.6, and the amino acid sequence of SuIL-15Rα-dFc-γ4 is shown in SEQ ID No.8. Show.

The above-mentioned mutations to the hinge region and Fc fragment of human IL-15 and human IgG4 are performed according to conventional mutation methods in the art.

The structure of the above-mentioned IL-15/SuIL-15Rα-dFc-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-dFc-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-dFc-γ4 and the expression vector pPICZα-IL-15 were inserted into Pichia pastoris 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-dFc-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-dFc-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 construction method of the above-mentioned IL-15/SuIL-15Rα-dFc-γ4 complex protein The nucleotide sequence of SuIL-15Rα-dFc-γ4 in .

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-dFc-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-dFc-γ4 complex protein.

After long-term research and accumulation of fusion proteins, the inventors constructed an IL-15/SuIL-15Rα-dFc-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. Acid is mutated to glutamic acid, and the 297th asparagine is mutated to glutamine to reduce, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The hinge region of IgG4 is mutated, and the 228th serine on the amino acid sequence is mutated to proline to prevent the formation of half-antibody molecules and make the disulfide bond pairing in the antibody molecule correctly, and stabilize the intrachain disulfide bond to obtain IgG4 Fc mutation. Then the IgG4 Fc variant was linked with the sushi domain of IL-15Rα through the hinge region in the IgG4Fc variant to obtain SuIL-15Rα-dFc-γ4, where dFc was a double-chain Fc, and finally expressed by Pichia pastoris The system produces the IL-15/SuIL-15Rα-dFc-γ4 complex protein; compared with the expensive and complex mammalian cell expression system, the Pichia pastoris system has low cost, no virus infection, and the recombinant protein is expressed in a secreted form, which is convenient for The invention has the advantages of high purification and high yield; the present invention completes the structural design of the complex protein, the construction of the vector, the establishment of the Pichia pastoris expression and purification system at the pilot level, and the related pharmacodynamics and pharmacokinetics research. , laying the foundation for its clinical trials and future applications.

Description of drawings

Fig. 1 is a schematic diagram of construction of an expression vector and a schematic diagram of the structure of the IL-15/SuIL-15Rα-dFc-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-dFc-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-dFc-γ4 complex protein accumulation during fermentation.

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

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

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

Figure 7 shows the in vivo biological activity detection results of IL-15/SuIL-15Rα-dFc-γ4 complex protein, wherein A is the photo and spleen weight of mice, 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 Pichiapastoris, 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-dFc-γ4 fusion protein expression vector

Amplification of 1SuIL-15Rα-dFc-γ4 Fusion Gene

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

1.2 Using pPICZα-Fc-mut as the template, use primer 3 and primer 4 to amplify the second target gene fragment. The PCR program is: 94°C for 5 min, (94°C for 30s, 55°C for 40s, 72°C for 50s) this program Set 32 cycles, 72°C for 10min;

1.3 Perform agarose gel electrophoresis on the amplified products in 1.1 and 1.2, and according to the operation steps of the DNA gel recovery kit, cut the gels corresponding to the two segments of the target gene and recover, that is, the first purpose is obtained. Gene fragment and the second target gene fragment;

1.4 Mix the first target gene fragment and the second target gene fragment as a template without adding primers for amplification. The PCR program is set to: 94°C for 5 min, (94°C for 30s, 55°C for 40s, 72°C for 60s). The segment program was set for 8 cycles, 72°C for 10 minutes; then primer 1 and primer 4 were added to it for amplification, and the PCR program was set to: 94°C for 5min, (94°C for 30s, 55°C for 40s, 72°C for 60s) 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-dFc-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 "pPICZα-Fc-mut" is a plasmid pPICZα containing IgG4 Fc variants mutated at positions 228, 235 and 297;

The function of pPIC9-SuIL-15R and pPICZα-Fc-mut is to connect the IgG4 Fc variant and the sushi domain of IL-15Rα through the hinge region in the human IgG4 Fc variant;

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 the expression vector of SuIL-15Rα-dFc-γ4 fusion protein

2.1 The expression vector pPIC9 and the gene SuIL-15Rα-dFc-γ4 obtained in step 1 were double digested using restriction enzymes Xho I and Not I; the digested product of SuIL-15Rα-dFc-γ4 was cleaned and recovered by PCR reagent Recovering the cassette, using DNA gel recovery kit to recover the digested vector pPIC9;

2.2 The enzyme digested product of SuIL-15Rα-dFc-γ4 was ligated with the digested vector pPIC9 using T4 DNA ligase to obtain the ligated product, and the ligated product was transformed into E. coli cells; the specific operation steps are: Add 10 μL of the ligated product to 100 μL of E. coli DH5α competent cells, let stand on ice, and mix gently every 10 min. After 30 min, heat shock the E. coli cells with the ligated product in a water bath ( 42°C, 90s), put it on ice immediately after completion, add 400μL of LB medium (no resistance) after 5min, put it on a shaker at 37°C for recovery for about 45min to 1h, and then take 50-100μL of bacterial suspension for coating It was placed on an ampicillin-resistant LB plate and placed upside down in a 37°C incubator for 12-16 hours. Add 3 mL of LB medium containing ampicillin resistance to the shaker tube, pick a single colony on the plate with a sterile small pipette tip or toothpick and clone it into the shaker tube, shake at 37°C, 220rpm for 6-8h, Then the bacterial liquid was amplified by PCR, and the fragment whose size was the same as expected was named pPIC9-SuIL-15Rα-dFc-γ4. The plasmid corresponding to the positive clone was extracted with a plasmid mini-extraction kit and sent to Shanghai Sangon Biotechnology for sequencing.

Experimental results: The plasmid sequencing results were consistent with the expected gene sequence, and the clone was constructed correctly.

Example 2 Yeast expression and purification of IL-15/SuIL-15Rα-dFc-γ4 complex protein

Pichia pastoris expression of complex 1 proteins

1.1 Gene expression and linearization: 50 mL of LB medium containing ampicillin resistance was added to the conical flask, and the positive clone pPIC9-SuIL-15Rα-dFc-γ4, plasmid pPICZα-IL-15 (as The plasmid pPICZα containing the IL-15 variant gene can be constructed according to conventional methods, 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 cell, so that the Pichia pastoris The IL-15 protein expressed in yeast cells was added to the medium by the inventor according to the conventional expression vector construction method, cultivated at 37°C for 12-16h, and the bacterial solution was lysed using a plasmid medium extraction kit to obtain the plasmid. (refer to the steps of the kit), obtain plasmid pPIC9-SuIL-15Rα-dFc-γ4 and plasmid pPICZα-IL-15; use endonuclease SalI and SacI to separate the extracted plasmid pPIC9-SuIL-15Rα-dFc-γ4 and plasmid pPICZα -IL-15 was linearized and recovered by ethanol precipitation. The concentration of the recovered product was determined by Nanodrop, and the concentration was adjusted between 0.5-1.0 μg/μL to obtain the linearized plasmid pPIC9-SuIL-15Rα-dFc-γ4 and the linearized plasmid pPICZα- IL-15;

1.2 Preparation of competent yeast cells: streak the cryopreserved Pichia GS115 on a YPD plate, and place it in a 30°C constant temperature incubator for cultivation; when the clone diameter reaches 1-2mm, use a small sterile pipette tip to pick a single line Clone, add it to a shaking tube containing 3-4 mL of YPD medium, and place it on a constant temperature shaker at 30°C for cultivation. After 24-48 hours, take out 1 mL of bacterial liquid and inoculate it into a conical flask containing 50 mL of YPD medium, and continue to cultivate , monitor the OD ₆₀₀ value of the bacterial solution until about 1-1.5, centrifuge at 1500g for 5 min at 4°C, discard the supernatant after centrifugation, resuspend with 40 mL of ice water, discard the supernatant after centrifugation, and repeat the washing with ice water; then add 20mL of 1M cold sorbitol was used to resuspend the pellet, centrifuged to discard the supernatant, and finally 500μL of 1M cold sorbitol was added to resuspend to obtain yeast competent cells;

1.3 Electroporation of pPIC9-SuIL-15Rα-dFc-γ4 into yeast cells: Add 100 μl of competent yeast cells to a sterile electroporation cup, and add 5-10 μg of linearized plasmid pPIC9-SuIL-15Rα-dFc-γ4 to mix with it, start Electroporation (the parameters of electroporation are set as: 2000V, 200Ω, 25μF), immediately add 1 mL of 1M cold sorbitol solution to the electroporation cup after the end, and after gently blowing and mixing, take an appropriate amount of bacterial liquid and spread it on the MD plate and place it at 30 Cultivated in a constant temperature yeast incubator. After the colonies appeared, use a sterile small pipette tip to pick multiple colony clones and add them to a shaking tube containing 4 mL of MGY medium, and place them on a yeast shaker for incubation at 30 °C for 18 minutes. After -24h, the bacterial solution was centrifuged at 12000g for 1 min, the supernatant was added to the loading buffer for sample preparation, and the expression of IgG4 Fc was detected by Western blot. 15Rα-dFc-γ4 yeast competent cells, and then 5-10 μg of the linearized plasmid pPICZα-IL-15 was introduced into yeast competent cells containing pPIC9-SuIL-15Rα-dFc-γ4 according to the above electrotransformation method, and the electroporated plasmid The bacterial liquid was coated on the YPD plate containing zeocin, and placed in a yeast shaker for constant temperature cultivation at 30 °C;

1.4 Screening of IL-15/SuIL-15Rα-dFc-γ4 positive expression clones: After the clones grow on the YPD plate in 1.3, pick multiple clones with a sterile small pipette tip and add them to shake bacteria containing 4 mL of YPD medium In the tube, incubate at 30°C for 24-48h on a constant temperature shaker, draw 3mL of bacterial liquid from it, centrifuge at 1500g for 5min, discard the supernatant, add 4mL BMMY medium to resuspend the pellet, place it in the shaker to continue culturing, and add more every 24h. Methanol solution (the final concentration of methanol is 1%), after 48 hours, centrifuge the bacterial solution at 12,000g for 1 min, draw the supernatant and prepare samples, conduct Dot-Blot preliminary screening, and select yeast strains with relatively high expression levels, and then the expression levels The relatively high yeast strains were spread on the YPD plate containing zeocin, and after the clones were grown, multiple clones were selected from them, and the bacteria were shaken and the supernatant was obtained. The antibody and anti-human IL-15 antibody were incubated to screen out high-expressing strains, and the remaining 1 mL of the corresponding strain was cryopreserved; this high-expressing strain was capable of expressing IL-15/SuIL-15Rα-dFc-γ4 complex Pichia strains of protein;

The above 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-dFc-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 9 to 33 express the IL-15/SuIL-15Rα-dFc-γ4 complex protein to varying degrees, from which the relatively high expression levels were selected. The strains numbered 15 and 16 were further screened and identified by Western Blot. Finally, the Pichia strain numbered 15-2 was selected as the Pichia pastoris that can highly express the IL-15/SuIL-15Rα-dFc-γ4 complex protein strains.

2Fermentation, purification and identification of complex proteins

2.1 Fermentation culture and complex protein expression of expression strains: Ferment the high expression strains screened in step 1, and the fermentation operation steps are carried out according to the fermentation guidelines of Invitrogen Company; inoculate the high expression strains into 4 mL of MGY medium, 30 ℃ constant temperature yeast shaker culture for 24-48h to obtain fermentation first-class seed solution; add 1mL first-class seed solution to 400mL BMGY medium, cultivate at 30 ℃ constant temperature shaker for 12-24h, monitor OD ₆₀₀ to 2-6, the Step to obtain fermentation secondary seed liquid; inoculate all secondary seed liquid into the fermenter of 6LBMGY medium, set the parameters of the fermenter as 28 ° C, pH 6.0, and start monitoring the dissolved oxygen and rotational speed in the fermenter, when When the dissolved oxygen in the tank rises rapidly, glycerol is added to the tank, and the wet weight of the bacteria is monitored, reaching about 180 g/L. Methanol is added to induce expression according to the three-step method. After the induction is completed, the pH value of the culture supernatant is adjusted. For 7-8, centrifuge at 10000g for 20min, collect the supernatant, and filter with 0.45μm and 500KDa microfilters successively;

2.2 Capture of complex proteins: Load the filtered supernatant in step 2.1 onto a HitrapMabSelect affinity chromatography column. The specific steps are: first rinse the column with deionized water (5 column volumes), and then use PBS buffer Rinse with PBS buffer (3 column volumes), and then load the supernatant into the sample. After loading, rinse with PBS buffer (3 column volumes). After washing, use the eluent to elute the target protein. The liquid formula is: 100mM sodium citrate/citric acid, pH=3.0, and finally add 1M Tris-HCl to the collected eluate to adjust the pH to neutrality to obtain a neutral eluate;

2.3 Complex protein purification: use AKTA PURE 25, add PBS buffer to balance the Superdex200 molecular sieve, then load the neutral eluate obtained in 2.2, rinse with PBS buffer after completion, and collect the eluted sample to obtain IL-15/ SuIL-15Rα-dFc-γ4 complex protein.

2.4 Test: In the fermentation process of the complex protein in step 2.1, the accumulation of the complex protein after methanol induction was started. The results are shown in Figure 3. Figure 3 shows the IL-15/SuIL-15Rα-dFc-γ4 complex protein during the fermentation process. Cumulative time point detection results; it can be seen from Figure 3 that with the prolongation of the induction time, the IL-15/SuIL-15Rα-dFc-γ4 complex protein continued to accumulate until about 45h.

2.5 Identification: SDS-PAGE and Western Blot identification of the complex protein obtained in step 2.3, the results are shown in Figure 4; Figure 4 is the SDS-PAGE and Western blot of the IL-15/SuIL-15Rα-dFc-γ4 complex protein The identification results, A is the identification of SDS-PAGE, and B is the identification of Western blot; it can be seen from Figure 4A that the protein purity of IL-15/SuIL-15Rα-dFc-γ4 complex can reach more than 90%, and it is mainly dimerized. The body form exists, which is consistent with the expectation; it can be seen from Figure 4B that the complex protein contains IL-15 components and Fc-γ4 components, and the resulting protein should be IL-15/SuIL-15Rα-dFc-γ4 complex protein.

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

1 Experimental method

The biological activity of IL-15/SuIL-15Rα-dFc-γ4 complex protein was detected by CTLL-2 cell proliferation assay Make it rapidly dissolved in the medium, add 5mL complete medium (complete medium formula: RPMI 1640 medium + 10% fetal bovine serum + 200IU/mL IL-2), centrifuge at 200g for 5min, discard the supernatant, add 1mL complete culture medium The cell pellets were resuspended and inoculated into a T25 culture flask containing complete medium for 2-3 days. When the cells were in good growth condition, a small amount of cell suspension was taken for counting. When the cell density reached 2×10 ⁵ cells/mL After passage, the cell suspension was gently mixed and added to a 15mL centrifuge tube, centrifuged at 200g for 5min, the supernatant was discarded, the cell pellet was resuspended in 1mL complete medium, and then inoculated at 1-2×10 ⁴ cells/mL after counting. , and then subculture according to the above-mentioned passage density and method;

1.2 Determination of the in vitro biological activity of IL-15/SuIL-15Rα-dFc-γ4 complex protein: Gently mix CTLL-2 cells in logarithmic growth phase, add them to a 15mL centrifuge tube, centrifuge at 200g for 5min, and discard the supernatant , resuspend the cells with 5 mL of basal medium (basal medium formula: RPMI-1640 medium + 10% FBS), discard the supernatant after centrifugation, and repeat the washing once; resuspend the pellet with 1 mL of basal medium and count, adjust The cell density was increased to 1.67×10 ⁵ cells/mL, and 60 μL of cell suspension (ie, 1×10 ⁴ cells/well) was added to each well of a 96-well cell culture plate; rhIL-15 and IL-15/SuIL-15Rα-dFc-γ4 complex protein, three replicate wells were set for each protein concentration, 40 μL of protein diluent was added to each well, and cultured for 48 h. After the incubation, 20 μL of 5 mg/mL MTT solution was added to each well. , placed in the incubator for 4 h, then centrifuged and discarded the supernatant, added 200 μL of DMSO to dissolve the formazan crystals, shaken on a shaker to fully dissolve the crystals, and used a microplate reader to measure the absorbance at OD ₄₉₀ nm.

2 Experimental results:

Using Graphpad Prism software, the detection results were fitted with a four-parameter logistic equation to obtain the EC ₅₀ value. The results are shown in Figure 5. Figure 5 shows the in vitro biological activity of the IL-15/SuIL-15Rα-dFc-γ4 complex protein Test results;

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-dFc-γ4 complex protein to stimulate the proliferation of CTLL-2 cells is about 5.99 pM. At 14.66 pM, the in vitro biological activity of the IL-15/SuIL-15Rα-dFc-γ4 complex protein was comparable to other reported IL-15 complexes.

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

1 Experimental method:

The healthy C57BL/6 mice aged 6-8 weeks were randomly divided into 2 groups, with 4 mice in each group. -15 moles of the same amount), the mice were injected into the tail vein, the tail was cut at 0.25, 0.5, 1, 2, and 4 hours after the injection, and 100 μL of blood from the rhIL-15 group was collected at 0.5, 2, 4, 8 , 12, 24, 48, and 72 hours, 100 μL of blood from the IL-15/SuIL-15Rα-dFc-γ4 group was collected, and the serum was collected by centrifugation. The protein concentration in serum was determined by ELISA, and the experimental results were processed by PKsolver software in a non-compartmental model to obtain the half-life value.

2 Experimental results:

The results are shown in Figure 6, which is the detection result of the circulating half-life in mice of IL-15/SuIL-15Rα-dFc-γ4 complex protein;

It can be seen from Figure 6 that the half-life of rhIL-15 is about 0.7h, and the half-life of IL-15/SuIL-15Rα-dFc-γ4 is about 14.62h, which indicates that IL-15/SuIL-15Rα-dFc-γ4 complexes The half-life of the somatic protein was significantly longer than that of commercial rhIL-15.

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

The healthy C57BL/6 mice aged 6-8 weeks were randomly divided into 3 groups with 5 mice in each group. The mice were given rhIL-15 0.28 mg/kg and IL-15/SuIL-15Rα-dFc-γ4 1 mg/kg to the mice. Tail vein injection was performed, and an equal volume of PBS buffer was injected into the control group. After 72 hours, the mice were killed by cervical dislocation, and the mouse spleen was removed and placed in pre-cooled PBS buffer; the mouse spleen was weighed. , and then the mouse spleen was prepared into a single-cell suspension using an automatic tissue disruptor, filtered through a nylon mesh, and centrifuged at 4°C, 2200 rpm for 10 min; the supernatant was discarded, and the red blood cell lysate was added to lyse the red blood cells, and used again after the end. Nylon mesh filtration, centrifugation (same centrifugal force as above), discard the supernatant, wash the cell pellet once with PBS buffer (3000rpm, 4°C, 5min), then count the cells, take 10 ⁴ cells/tube for flow detection operation, use The PBS buffer containing 10% rat serum was blocked at 4°C for 30min, and then antibody labeling was performed at 4°C for 1h (labeled molecules include CD3, CD8, CD4, CD44, NK1.1, CD19, CD45), and PBS buffer was used after the end. Wash twice, and use flow cytometer to detect the expression of each surface molecule;

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

As can be seen from Figure 7A, compared with the control group (PBS group) and rhIL-15 group, the spleen weight of mice injected with IL-15/SuIL-15Rα-dFc-γ4 complex protein increased significantly, indicating that the immune system in the spleen increased significantly. Cells proliferated significantly; as can be seen from Figure 7B, flow analysis showed that compared with the PBS group and the rhIL-15 group, in the spleen of the mice in the IL-15/SuIL-15Rα-dFc-γ4 group, CD8 ⁺ T cells , CD8 ⁺ CD44 ⁺ T cells, NK cells, NKT cells and the number of cells increased significantly; the above results show that the activity of IL-15/SuIL-15Rα-dFc-γ4 complex protein in inducing immune cell proliferation is significantly better than that of single body IL-15 molecule.

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-dFc-γ4 complex protein and its construction method and application

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Claims (8)

1. An IL-15/SuIL-15R alpha-dFc-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-dFc- 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 the hinge region and the Fc fragment of human IgG4 to obtain an IgG4Fc variant with the nucleotide sequence shown as SEQ ID No. 5;

s3, connecting the IgG4Fc variant with the sushi structure domain of IL-15R alpha through a hinge region in the IgG4Fc variant to obtain SuIL-15R alpha-dFc-gamma 4, wherein the nucleotide sequence of the SuIL-15R alpha-dFc-gamma 4 is shown as SEQ ID No. 7;

s4, co-expressing the IL-15 variant and SuIL-15R alpha-dFc-gamma 4 to obtain the IL-15/SuIL-15R alpha-dFc-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-dFc- 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-dFc-y 4 in the method of constructing the IL-15/sull-15 ra-dFc-y 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-15R alpha-dFc-gamma 4 complex protein as defined in claim 1 in preparing antiviral and antitumor drugs.

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

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