CN111690069B

CN111690069B - IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein and construction method and application thereof

Info

Publication number: CN111690069B
Application number: CN201911105976.4A
Authority: CN
Inventors: 肖卫华; 徐欢; 郭雨刚; 邬婧; 邵长胜; 李�浩
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2019-11-13
Filing date: 2019-11-13
Publication date: 2022-04-19
Anticipated expiration: 2039-11-13
Also published as: CN111690069A

Abstract

The invention discloses an IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein, which comprises an amino acid sequence shown in SEQ ID No.1 or an amino acid sequence with more than 90% of identity with the SEQ ID No. 1. The invention also discloses a construction method of the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein. The invention also discloses two expression vectors. The invention also discloses a yeast strain. The invention also discloses application of the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein in preparing antiviral and antitumor drugs. The invention also discloses application of the expression vector or yeast strain in expression of IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein. The complex protein significantly prolongs the half-life of IL-15.

Description

IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein and construction method and application thereof

Technical Field

The invention relates to the technical field of protein engineering, in particular to an IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein, a construction method and application thereof.

Background

Interleukin 15(IL-15) is an important immunologically active protein that has been used clinically in antiviral and antitumor therapeutic studies. However, the monomer IL-15 has low molecular potency and short half-life period, which severely limits the application of the monomer IL-15 as a clinical drug. In order to improve the therapeutic effect of IL-15, researchers have developed several IL-15 complexes one after the other, e.g., as early as 2006, researchers such as Erwan Mortier connect IL-15 to the sushi domain of IL-15 receptor alpha to form complex RLI, which results in a significant increase in IL-15 activity.

In addition to forming complexes with receptors, fusion to the Fc portion of antibodies is an important method for extending the half-life of protein drugs. There have been several successful cases in this regard. For example, the globally marketed Fc fusion protein Enbrel (TNFR-Fc) and the FDA recently approved fusion drug protein of recombinant blood VIII factor and Fc developed by baijiandi corporation on the market. There have also been several 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-15 Ra-IgG 1-Fc, and in 2011 ALtor Bioscience developed ALT-803 (now more known as N-803). Both effectively increase the in vivo half-life of IL-15.

Fc fusion proteins extend plasma half-life in vivo via neonatal Fc receptor (FcRn). The specific mechanism is that the Fc fusion protein is combined with FcRn of phagocytic cells in vivo and is finally circularly released to the outside of the cells so as to avoid lysosome degradation.

Disclosure of Invention

Based on the technical problems in the background art, the invention provides an IL-15/SuIL-15 Ra-mFc-Gamma 4 complex protein, a construction method and application thereof, the invention constructs the IL-15/SuIL-15 Ra-mFc-Gamma 4 complex protein based on Pichia pastoris, and the half-life period of IL-15 is obviously prolonged.

The IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein provided by the invention comprises an amino acid sequence shown in SEQ ID No.1 or an amino acid sequence which has more than 90% of identity with the SEQ ID No. 1.

The amino acid sequence shown as SEQ ID No.1 is shown as SEQ ID No. 2.

The invention also provides a construction method of the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein, which comprises the following steps:

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.

The amino acid sequence of the IL-15 variant is shown as SEQ ID No.4, the amino acid sequence of the Fc variant is shown as SEQ ID No.6, the amino acid sequence of the connecting peptide is shown as SEQ ID No.8, and the amino acid sequence of the SuIL-15R alpha-mFc-gamma 4 is shown as SEQ ID No. 10.

The above-described mutation of the Fc fragment of human IL-15 or human IgG4 was carried out according to a conventional mutagenesis method in the art.

The structure of the IL-15/SuIL-15 Ra-mFc-Gamma 4 complex protein and the construction method thereof are schematically shown in FIG. 1, FIG. 1 is a schematic diagram of the construction and structure of an expression vector of the IL-15/SuIL-15 Ra-mFc-Gamma 4 complex protein, wherein A is a schematic diagram of the construction of the expression vector; b is a structural schematic diagram. In FIG. 1A, an expression vector pPIC9-SuIL-15 Ra-mFc-Gamma 4 and an expression vector pPICZ alpha-IL-15 are respectively inserted into corresponding sites in the genome of a Pichia pastoris GS115 strain in a homologous recombination mode by utilizing a HIS4 sequence and a 5' AOX1 sequence on the vectors, so that the Pichia pastoris strain capable of expressing IL-15/SuIL-15 Ra-mFc-Gamma 4 complex protein is constructed.

The invention also provides an expression vector which comprises a 5' AOX1 promoter, a transcription terminator, an antibiotic resistance gene and a secretion signal peptide, and also comprises the nucleotide sequence of the IL-15 variant in the construction method of the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein.

The invention also provides an expression vector, which comprises a 5' AOX1 promoter, a transcription terminator, an antibiotic resistance gene and a secretion signal peptide, and also comprises a nucleotide sequence of the SuIL-15 Ra-mFc-Gamma 4 in the construction method of the IL-15/SuIL-15 Ra-mFc-Gamma 4 complex protein.

The invention also provides a yeast strain, which comprises the two expression vectors.

Preferably, the yeast strain is a pichia strain.

The invention also provides application of the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein in preparing antiviral and antitumor drugs.

The invention also provides application of the expression vector or the yeast strain in expression of the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein.

The inventor constructs the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein based on Pichia pastoris through long-term fusion protein research and accumulation, and obviously prolongs the half-life period of IL-15; the method comprises the steps of mutating IL-15, mutating asparagine at the 72 th position on an IL-15 amino acid sequence into aspartic acid so as to improve the bioactivity of the IL-15, and mutating asparagine at the 71 th position, the 79 th position and the 112 th position into glutamine so as to reduce immunogenicity possibly brought by potential glycosylation modification to obtain an IL-15 variant; mutating the Fc fragment of human IgG4 to change leucine at position 235 to glutamic acid and asparagine at position 297 to glutamine in the amino acid sequence of the Fc fragment, thereby reducing, for example, antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to obtain an Fc variant; the activity of the IL-15/SuIL-15 Ra-mFc-gamma 4 complex protein can be maintained by selecting a suitable connecting peptide to connect the Fc variant with the sushi domain of IL-15 Ra to obtain SuIL-15 Ra-mFc-gamma 4, wherein mFc is single-chain Fc, and selecting a GS connecting peptide with flexibility; the molecular weight of the single-chain Fc is smaller, and the single-chain Fc can more easily enter tumor tissues or infected tissues in vivo to play a better treatment effect; the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein based on the antibody single-chain Fc is produced by adopting a simple and cheap pichia pastoris expression system; the Chinese horizontal production process is established, pharmacodynamics and pharmacokinetics research are carried out, and a foundation is laid for future clinical tests and application of the Chinese horizontal production process.

Drawings

FIG. 1 is a schematic diagram of the construction and structure of an expression vector for IL-15/SuIL-15R α -mFc- γ 4 complex protein, wherein A is a schematic diagram of the construction of the expression vector; b is a structural schematic diagram.

FIG. 2 shows the results of screening and identification of Pichia pastoris strains expressing IL-15/SuIL-15R alpha-mFc-gamma 4 complex proteins, wherein A is Dot-Blot screening result and B is Western Blot identification result.

FIG. 3 shows the results of detection of accumulation time points of IL-15/SuIL-15R α -mFc- γ 4 complex proteins during fermentation.

FIG. 4 shows the SDS-PAGE and Western Blot identification of IL-15/SuIL-15R α -mFc- γ 4 complex protein, wherein A is the SDS-PAGE identification and B is the Western Blot identification.

FIG. 5 shows the results of in vitro bioactivity assay of IL-15/SuIL-15R α -mFc- γ 4 complex protein.

FIG. 6 shows the results of the in vivo circulating half-life test of IL-15/SuIL-15R α -mFc- γ 4 complex protein in mice.

FIG. 7 shows the results of in vivo measurement of the bioactivity of IL-15/SuIL-15R α -mFc- γ 4 complex protein, wherein A is a photograph of mouse spleen and the weight of spleen, and B is the ratio and number of the mouse spleen immune cell subsets.

Detailed Description

The technical solution of the present invention will be described in detail below with reference to specific examples.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

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

Experimental animals: experimental C57BL/6 mice were bred and housed in SPF grade housing, the institute of Life sciences, university of science and technology.

Experimental reagent: DNA gel recovery kit (Axygen), PCR clean recovery kit (Axygen), plasmid miniprep kit (Axygen), anti-human IgG4Fc antibody (Abcam), anti-human IL-15 monoclonal antibody (Dr. Ded. bioengineering Co., Ltd.), anti-rabbit secondary antibody (Biolegend), RPMI 1640 medium (HyClone), fetal bovine serum (BI), IL-2 (Jiangsu Jinli pharmaceutical industry Co., Ltd.), rhIL-15 (PeproTech), IL-15 capture antibody and IL-15 detection antibody (R & D Systems).

The main equipment comprises: ultraviolet spectrophotometric analyzer (SHIMADZU, japan), microplate reader (Bio Tek, usa), fully automatic tissue disruptor (Miltenyi Biotec, germany), flow cytometer (Beckman Coulter, usa), cell counter (Countstar, china).

Example 1 SuIL-15R α -mFc- γ 4 fusion protein expression vector construction

Amplification of 1 SuIL-15R alpha-mFc-gamma 4 fusion Gene

1.1 Using pPIC9-SuIL-15R as template, using primer 1 and primer 2 to amplify fragment 1, the PCR procedure was: 94 ℃ for 5min, (94 ℃ for 30s, 55 ℃ for 40s, 72 ℃ for 20s) the program set up 32 cycles, 72 ℃ for 10 min;

1.2 using pPICZ alpha-Fc-mut as template, using primer 3 and primer 4 to amplify fragment 2, the PCR procedure was: 94 ℃ for 5min, (94 ℃ for 30s, 55 ℃ for 40s, 72 ℃ for 50s) the program set up 32 cycles, 72 ℃ for 10 min;

1.3, performing agarose gel electrophoresis on the products amplified in the steps 1.1 and 1.2, and recovering two sections of gene fragments, namely the fragment 1 and the fragment 2, by using a DNA gel recovery kit (operating according to the kit specification) after electrophoresis;

1.4 fragment 1 and fragment 2 were mixed as template and amplified without primer addition, and the PCR program was set as: 94 ℃ for 5min, (94 ℃ for 30s, 55 ℃ for 40s, 72 ℃ for 60s) this program set up 8 cycles, 72 ℃ for 10 min; then adding primer 1 and primer 4 for amplification, and setting the PCR program as follows: 94 ℃ for 5min, (94 ℃ for 30s, 55 ℃ for 40s, 72 ℃ for 60s) the program set up 32 cycles, 72 ℃ for 10 min; carrying out agarose gel electrophoresis on the amplification product, and then recovering a section of gene by adopting a DNA gel recovery kit, wherein the two ends of the gene are provided with Xho I and Not I enzyme cutting sites and are named as SuIL-15R alpha-mFc-gamma 4;

the sequences of the primers 1 to 4 are shown in the following table:

the "pPIC 9-SuIL-15R" is a plasmid pPIC9 containing a SuIL-15R gene, and the SuIL-15R is a sushi structural domain gene of IL-15 Ra;

the "pPICZ alpha-Fc-mut" is a plasmid pPICZ alpha containing IgG4Fc gene with mutations at the 228 th site, the 235 th site and the 297 th site;

pPIC9-SuIL-15R, pPICZ α -Fc-mut functions to link the Fc variant to the sushi domain of IL-15 Ra via a linker peptide, wherein the 228-site mutation is not linked by the linker peptide to the sequence of the SuIL-15 Ra-mFc- γ 4;

the pPIC9-SuIL-15R, pPICZ alpha-Fc-mut is obtained by constructing genes into corresponding plasmids by the inventor according to a conventional expression vector construction method.

Construction of Pichia pastoris expression vector of 2 SuIL-15R alpha-mFc-gamma 4 fusion protein

2.1 carrying out double enzyme digestion on the gene SuIL-15R alpha-mFc-gamma 4 obtained in the step 1 and the purchased plasmid pPIC9 vector by using restriction enzymes Xho I and Not I; then, recovering the gene SuIL-15R alpha-mFc-gamma 4 subjected to enzyme digestion by using a PCR clean recovery kit, and recovering a plasmid pPIC9 vector subjected to enzyme digestion by using a DNA gel recovery kit;

2.2 connecting the enzyme-cut gene SuIL-15R alpha-mFc-gamma 4 with the enzyme-cut plasmid pPIC9 vector by using T4 DNA ligase to obtain a connected product; adding 10 mul of the connected product into 100 mul of escherichia coli DH5 alpha competent cells, standing on ice for 30min, flicking and uniformly mixing every 10min, then performing heat shock at 42 ℃ for 90s in a water bath kettle, taking out, immediately standing on ice for 3-5min, adding 400 mul of sterile LB liquid medium (non-resistant), performing shake culture at 37 ℃ for 45min, coating 100 mul of bacterial suspension on a LA medium plate (ampicillin-resistant LB plate), and placing in a 37 ℃ incubator for culture; after the colony grows out from the plate, selecting a single colony to clone into a bacteria shaking tube containing LA liquid culture medium (LB culture medium with ampicillin resistance), culturing for 6-8h at 37 ℃, then carrying out PCR identification by taking bacterial liquid as a template, obtaining a positive clone named pPIC9-SuIL-15R alpha-mFc-gamma 4, extracting the bacterial liquid corresponding to the positive clone by using a plasmid small-amount extraction kit, and carrying out marine organism sequencing.

The experimental results are as follows: the sequencing result is consistent with the theoretical expected result, and the cloning construction is correct.

Example 2 expression and purification of IL-15/SuIL-15R α -mFc- γ 4 Complex proteins

1 expression of Complex proteins

1.1 Gene expression and linearization: the positive clone pPIC9-SuIL-15R alpha-mFc-gamma 4 and the plasmid pPICZ alpha-IL-15 (the plasmid pPICZ alpha containing the IL-15 variant gene can be constructed by a conventional method, the function of the plasmid pPICZ alpha-IL-15 is to integrate the IL-15 variant gene into a corresponding site in a pichia pastoris cell by using a 5' AOX1 sequence so as to express the IL-15 protein in the pichia pastoris cell, and the plasmid is extracted by using a plasmid medium-volume extraction kit to obtain an expression plasmid pPIC9-SuIL-15R alpha-mFc-gamma 4 and an expression plasmid pPICZ alpha-IL-15; linearizing an expression plasmid pPIC9-SuIL-15R alpha-mFc-gamma 4 and an expression plasmid pPICZ alpha-IL-15 by using endonucleases SalI and SacI respectively, and performing ethanol precipitation and recovery; the specific operation steps of recovery are as follows: adding 1/10 volume of 3M sodium acetate (pH 5.2) into the enzyme digestion reaction system, mixing well, adding 2-2.5 times volume of glacial ethanol, mixing well, and precipitating in a refrigerator at-20 deg.C for 1-2 h; centrifuging at 4 ℃ for 10min at 12000g, discarding the supernatant, adding 1mL of 70% ethanol for heavy suspension precipitation, centrifuging at 4 ℃ for 10min at 12000g, discarding the supernatant, and repeatedly washing once; opening an EP tube cover at room temperature to completely volatilize residual ethanol, adding a proper amount of deionized water for dissolution, measuring the concentration by using Nanodrop, and regulating the concentration to be 0.5-1.0 mu g/mu L to obtain a linearized plasmid pPIC9-SuIL-15R alpha-mFc-gamma 4 and a linearized plasmid pPICZ alpha-IL-15;

1.2 preparation of Yeast competence: taking out the frozen GS115 Pichia pastoris strain from the refrigerator, streaking on a YPD plate, and culturing in a constant temperature incubator at 30 ℃; observing yeast clone growth to about 1-2mm in diameter, selecting single clone in 3-4mL YPD culture medium, culturing at 30 deg.C in yeast shaker for 24-48 hr, inoculating 1mL bacterial liquid into 50mL YPD culture medium, culturing at 30 deg.C in yeast shaker, monitoring OD value of bacterial liquid, and waiting for OD value₆₀₀Transferring the bacterial liquid to a 50mL centrifuge tube, centrifuging at 4 deg.C and 1500g for 5min, and separatingDiscarding the supernatant after the heart, adding 40mL of ice water for resuspension, centrifuging at 4 ℃ for 5min at 1500g, discarding the supernatant, and repeatedly washing with ice water once; resuspending the centrifuged precipitate with 20mL of 1M sorbitol, centrifuging at 4 deg.C for 5min at 1500g, discarding the supernatant, and finally adding 500 μ L of 1M sorbitol to resuspend the thallus to obtain yeast competent cells;

1.3 electrotransformation of pPIC9-SuIL-15R α -mFc- γ 4: 10 μ L of linearized plasmid pPIC9-SuIL-15R α -mFc- γ 4(5-10 μ g) was mixed with 100 μ L of yeast competent cells and added to a sterile, pre-cooled cuvette; the electrotransport device parameters are set as follows: 2000V, 200 Ω, 25 μ F; starting electric transfer, immediately adding 1mL of 1M cold sorbitol into an electric transfer cup after the electric transfer is finished, slightly and uniformly mixing, coating a bacterial liquid on an MD (MD) flat plate, and culturing the flat plate in a constant-temperature yeast incubator at 30 ℃ until bacterial colonies appear; selecting a plurality of clones to 4 mM MgGY culture medium, culturing for 18-24h at constant temperature of 30 ℃ in a yeast shaker, taking bacterial liquid, centrifuging for 1min at 12000g, taking supernate, carrying out Western Blot detection by using an anti-human IgG4Fc antibody, and preparing the obtained positive clones into yeast competent cells containing pPIC9-SuIL-15R alpha-mFc-gamma 4 according to a yeast competence preparation method in 1.2; then 10. mu.l of linearized plasmid pPICZ alpha-IL-15 was introduced into yeast competent cells containing pPIC9-SuIL-15R alpha-mFc-gamma 4 according to the above electrotransformation method, and the electrotransformed bacterial solution was spread on YPD plates (containing zeocin);

1.4 screening of IL-15/SuIL-15R α -mFc- γ 4 Positive expression clones: placing YPD plates (containing zeocin) coated with bacterial liquid in 1.3 in a 30 ℃ constant temperature yeast incubator for culture until bacterial colonies appear, selecting a plurality of clones to 4mL YPD culture medium, culturing the YPD plates at 30 ℃ for 24-48h by a yeast shaker, sucking 3mL bacterial liquid from the YPD plates, centrifuging for 5min at 1500g, discarding supernatant, adding 4mL BMMY culture medium for heavy suspension and precipitation, placing the YPD plates in a shaker for continuous culture, supplementing methanol solution (the final concentration of methanol is 1%) every 24h, centrifuging for 1min at 12000g of bacterial liquid after 48h, sucking supernatant and preparing samples, performing Dot-Blot primary screening to select yeast strains with relatively high expression quantity, coating the yeast strains with relatively high expression quantity on the YPD plates containing zeocin, selecting a plurality of clones from the YPD plates according to the mode after the clones grow out, shaking the bacterial liquids and obtaining supernatant for Blot identification, and using 4 anti-Fc antibody and anti-human IL-15 antibody respectively, screening out high-expression strains, and freezing and preserving the remaining 1mL of bacterial liquid of the corresponding strains; the high expression strain is a pichia pastoris strain capable of expressing IL-15/SuIL-15R alpha-dFc-gamma 4 complex protein;

the Dot-Blot and Western Blot identification results are shown in FIG. 2, and FIG. 2 is a screening and identification result of a Pichia pastoris strain capable of expressing IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein, wherein A is a Dot-Blot screening result and B is a Western Blot identification result.

As can be seen from fig. 2: the Dot-Blot initial screening result shows that Pichia pastoris strains with numbers from 24 to 68 express IL-15/SuIL-15R alpha-mFc-gamma 4 complex proteins to different degrees, strains with numbers from 44 and 62 with relatively high expression levels are selected for further plating screening, Western Blot is used for identification, and the Pichia pastoris strain with number from 62-4 is finally selected as the Pichia pastoris strain capable of highly expressing the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein.

2 fermentation, purification and identification of complex protein

2.1 fermentation of Complex proteins: fermenting the high-expression strain screened in the step 1, wherein the fermentation operation steps are carried out according to fermentation guidelines of Invitrogen company; inoculating an expression strain in 4mL of MGY culture medium, and carrying out shake culture on constant-temperature yeast at 30 ℃ for 24-48h to obtain a bacterial liquid, namely a fermentation primary seed liquid; inoculating 1mL of the first-stage seed solution into 400mL of BMGY culture medium, and performing shake culture at a constant temperature of 30 ℃ for 12-24h until OD600 is 2-6, wherein the obtained bacterial solution is a fermentation second-stage seed solution; inoculating 400mL of secondary seed liquid into a fermentation tank containing a BMGY culture medium, setting the parameters of the fermentation tank to 28 ℃ and pH 6.0, synchronously monitoring dissolved oxygen and rotation speed in the fermentation tank, starting to add glycerol when the dissolved oxygen in the tank rises rapidly, monitoring the wet weight of thalli, starting to add methanol for induction according to a three-step method recommended by Invitrogen company, after induction is finished, adjusting the pH value of a culture supernatant to 7-8, centrifuging for 20min at 10000g, collecting the supernatant, and filtering by using microfiltration cylinders of 0.45 mu m and 500KDa successively;

2.2, purification: and (3) loading the filtered supernatant obtained in the step 2.1 to a Hitrap MabSelect of a parental chromatographic column, which comprises the following specific steps: washing the chromatographic column by using deionized water with 5 column volumes, washing the chromatographic column by using PBS buffer solution with 3 column volumes, then passing the supernatant through the column, washing the chromatographic column by using the PBS buffer solution with 3 column volumes after the column passing is finished, eluting the target protein by using eluent, wherein the formula of the eluent is as follows: 100mM sodium citrate/citric acid, pH 3.0, after the elution is finished, adding 1M Tris-HCl into the collected eluent to adjust the pH to be neutral to obtain neutral eluent; then fine purification is carried out, and the specific steps are as follows: and (3) using AKTAPURE 25, balancing the Superdex200 molecular sieve by using a PBS (phosphate buffer solution), loading a neutral eluent after balancing, washing by using the PBS buffer solution, and collecting an eluted sample to obtain the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein.

2.3 test: monitoring the accumulation amount of the complex protein in the methanol induction in the fermentation process of the complex protein in the step 2.1, wherein the result is shown in figure 3, and figure 3 is a detection result of the accumulation time point of the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein in the fermentation process; as can be seen in FIG. 3, the accumulation of IL-15/SuIL-15R α -mFc- γ 4 complex protein continued to increase during the induction period of 0-32 h; when the induction time reaches 43h, the accumulation amount of IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein is not increased any more, and is even reduced.

2.4 identification: performing SDS-PAGE and Western Blot identification on the complex protein obtained in the step 2.2, and obtaining a result shown in figure 4; FIG. 4 shows the SDS-PAGE and Western Blot identification results of IL-15/SuIL-15R α -mFc- γ 4 complex protein, wherein A is the SDS-PAGE identification and B is the Western Blot identification; from FIG. 4A, it can be seen that the IL-15/SuIL-15R α -mFc- γ 4 complex protein is more than 90% pure and exists mainly in monomeric form, consistent with expectations; as can be seen from FIG. 4B, the complex protein contains an IL-15 component and an Fc- γ 4 component, and the resulting recombinant protein should be an IL-15/SuIL-15R α -mFc- γ 4 complex protein.

Example 3 in vitro bioactivity assay of IL-15/SuIL-15R α -mFc- γ 4 Complex proteins

1 method of experiment

Determination of the cell proliferation-promoting Activity of IL-15/SuIL-15R α -mFc- γ 4 Complex protein Using CTLL-2 cells

1.1 CTLL-2 cell recovery and passage: taking out CTLL-2 cells (as a gift from the institute of immunology, institute of bioscience, university of science and technology, China), rapidly melted by shaking in a water bath at 37 deg.C, adding into a centrifuge tube containing 5mL of complete medium (the formula of complete medium is RPMI 1640 medium + 10% fetal calf serum (BI company) +200IU/mL IL-2), centrifuging at room temperature for 5min at 200g, resuspending with 1mL of complete medium after centrifugation, counting, and counting at 4 × 10⁴cells/mL at 25cm²A culture bottle; when the cell density reaches 2X 10⁵Passage is carried out when cells/mL, a 15mL centrifuge tube is taken, the cell suspension is added into the centrifuge tube, the centrifuge tube is centrifuged for 5min at room temperature at 200g, after the centrifugation is finished, the cell suspension is resuspended by 1mL complete culture medium, the counting is carried out, and the cell suspension is counted according to the 1-2 × 10⁴cells/mL, after which the cells were passaged every 2-3 days at the above cell density;

1.2 determination of the cell proliferation-promoting Activity of the IL-15/SuIL-15R α -mFc- γ 4 Complex protein: centrifuging CTLL-2 cells cultured to logarithmic phase at room temperature at 200g for 5min, discarding the supernatant after centrifugation is finished, adding 5mL of RPMI-1640 culture medium to resuspend the cells, centrifuging at room temperature at 200g for 5min, and repeatedly washing twice; resuspending with 1mL of basal medium (basal medium formulation: RPMI-1640 medium + 10% FBS), counting, adjusting cell density, adding 60 μ L of cell suspension into 96-well plate to make cell number 1.0 × 10⁴cells/well; diluting rhIL-15 and IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein with a basic culture medium according to 2-fold gradient, adding 40 mu L of each well, culturing for 48h, developing by adopting MTT method after the culture is finished, and determining OD (optical density) by using an enzyme labeling instrument₄₉₀Absorbance at nm.

2, experimental results:

calculating the MTT colorimetric result by adopting Graphpad Prism software, performing curve fitting by using a four-parameter logistic equation, and calculating EC₅₀Values, results see FIG. 5, FIG. 5 shows the results of in vitro bioactivity assays for IL-15/SuIL-15R α -mFc- γ 4 complex proteins;

it can be seen from FIG. 5 that rhIL-15 stimulates the proliferation of CTLL-2 cells by EC₅₀EC with a value of about 5.99pM for stimulation of CTLL-2 cell proliferation by IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein₅₀Activity of IL-15/SuIL-15R α -mFc- γ 4 complex protein with a value of about 12.74pMComparable to other reported IL-15 complexes, and even more superior in activity.

Example 4 in vivo half-Life detection of IL-15/SuIL-15R α -mFc- γ 4 Complex proteins

1 experimental method:

selecting healthy C57BL/6 mice with the weight of 18-25g and the week period of 6-8, randomly dividing the mice into 2 groups, randomly dividing 4 mice in each group, giving the mice tail vein injection according to the principle that the molar weight of the injected IL-15 molecules is the same, namely rhIL-150.28 mg/kg and the dose of IL-15/SuIL-15R alpha-mFc-gamma 41 mg/kg, and cutting tails and taking blood at different time points, wherein the blood taking time points of the rhIL-15 mice are 0.25, 0.5, 1, 2 and 4h, and the blood taking time points of the IL-15/SuIL-15R alpha-mFc-gamma 4 mice are as follows: 0.5, 2, 4, 8, 10, 12, 24 and 48 hours, taking blood, centrifuging and collecting serum, measuring the protein concentration in a serum sample by adopting ELISA, and processing drug-substituted data by using PKsolver software according to a non-compartmental model.

2, experimental results:

the half-life of commercial rhIL-15 and IL-15/SuIL-15R alpha-mFc-gamma 4 complex was determined using C57BL/6 mice, as shown in FIG. 6, and FIG. 6 is the half-life test result of IL-15/SuIL-15R alpha-mFc-gamma 4 complex in vivo circulation in mice;

as can be seen from FIG. 6, the half-life of rhIL-15 was about 0.7h, the half-life of IL-15/SuIL-15 Ra-mFc- γ 4 was about 9.16h, and the half-life of the IL-15/SuIL-15 Ra-mFc- γ 4 complex protein was significantly longer than that of the commercial rhIL-15.

Example 5 in vivo bioactivity assay of IL-15/SuIL-15R α -mFc- γ 4 Complex proteins

Healthy C57BL/6 mice with the weight of 18-25g and the week period of 6-8 are selected and randomly divided into 3 groups, 5 mice are given to the mice in tail vein injection according to the dose of rhIL-150.28 mg/kg and IL-15/SuIL-15R alpha-mFc-gamma 41 mg/kg, the mice are injected with PBS buffer solution with the same volume in the control group, the eyeballs are picked and blood is taken after 72h, then the mice are killed by the neck breaking method, the spleens of the mice are picked and placed in the ice-cold PBS buffer solution, the spleens of the mice are photographed and weighed, and then the spleens are further processed for flow detection, and the specific processing mode is as follows:

grinding the spleen of the mouse by using a full-automatic tissue crusher, filtering by using a 100-mesh nylon net after grinding, centrifuging at 4 ℃ and 2200rpm for 10min after filtering; discarding the supernatant, dispersing the cells, adding 1mL of erythrocyte lysate, mixing uniformly, performing lysis for 5min at room temperature in a dark place until the cells are clarified, adding 10mL of PBS buffer solution to terminate the lysis, filtering the cells into a 15mL centrifuge tube through a 200-mesh nylon net, centrifuging the supernatant, adding 1mL of PBS buffer solution to wash the cells, centrifuging the cells at 4 ℃ and 3000rpm for 5min, performing resuspension by using 1 XPBS buffer solution containing 10% rat serum, sealing the cells in a dark place for 30min at 4 ℃, simultaneously diluting part of the cells, counting the cells by using a cell counter, performing flow antibody labeling on molecules CD3, CD8, CD4, CD44, NK1.1, CD19 and CD45 (purchased from Biolegend), sealing the cells for 1h at 4 ℃, washing the cells by using 1 XPBS buffer solution, centrifuging the cells, adding the 1 XPBS buffer solution to perform resuspension, and performing detection on a machine;

the detection results are shown in FIG. 7, and FIG. 7 is the detection results of the in vivo biological activity of IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein, wherein A is the mouse spleen picture and the spleen weight, and B is the ratio and number of the mouse spleen immune cell subsets;

as can be seen from FIG. 7A, the spleen of the mice injected with the IL-15/SuIL-15R α -mFc- γ 4 complex protein was significantly increased and the weight of the spleen was significantly increased as compared to the control group (PBS group) and the commercial rhIL-15 group; as can be seen from FIG. 7B, CD8 was found in spleen of mice injected with IL-15/SuIL-15R α -mFc- γ 4 complex protein⁺T cell, CD8⁺CD44⁺The proportion and the cell number of T cells (T cells with memory phenotype), NK cells and NKT cells are obviously increased compared with those of a control group (PBS group) and a commercial rhIL-15 group; the results show that the in vivo bioactivity of the IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein is obviously superior to that of a monomer IL-15 molecule.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Sequence listing

<110> university of science and technology in China

<120> IL-15/SuIL-15R alpha-mFc-gamma 4 complex protein, and construction method and application thereof

<130> 2019

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 412

<212> PRT

<213> Artificial Synthesis ()

<400> 1

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

405 410

<210> 2

<211> 1239

<212> DNA

<213> Artificial Synthesis ()

<400> 2

aactgggtga atgtaataag tgatttgaaa aaaattgaag atcttattca atctatgcat 60

attgatgcta ctttatatac ggaaagtgat gttcacccca gttgcaaagt aacagcaatg 120

aagtgctttc tcttggagtt acaagttatt tcacttgagt ccggagatgc aagtattcat 180

gatacagtag aaaatctgat catcctagca caagatagtt tgtcttctaa tgggcaagta 240

acagaatctg gatgcaaaga atgtgaggaa ctggaggaaa aaaatattaa agaatttttg 300

cagagttttg tacatattgt ccaaatgttc atccaaactt ctatcacgtg ccctcccccc 360

atgtccgtgg aacacgcaga catctgggtc aagagctaca gcttgtactc cagggagcgg 420

tacatttgta actctggttt caagcgtaaa gccggcacgt ccagcctgac ggagtgcgtg 480

ttgaacaagg ccacgaatgt cgcccactgg acaaccccca gtctcaaatg cattagagac 540

ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttctgcacc tgagttcgaa 600

gggggaccat cagtcttcct gttcccccca aaacccaagg acactctcat gatctcccgg 660

acccctgagg tcacgtgcgt ggtggtggac gtgagccagg aagaccccga ggtccagttc 720

aactggtacg tggatggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 780

ttccaaagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaac 840

ggcaaggagt acaagtgcaa ggtctccaac aaaggcctcc cgtcctccat cgagaaaacc 900

atctccaaag ccaaagggca gccccgagag ccacaggtgt acaccctgcc cccatcccag 960

gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctaccccagc 1020

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1080

cccgtgctgg actccgacgg ctccttcttc ctctacagca gactaaccgt ggacaagagc 1140

aggtggcagg aggggaatgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1200

tacacacaga agagcctctc cctgtctctg ggtaaataa 1239

<210> 3

<211> 342

<212> DNA

<213> Artificial Synthesis ()

<400> 3

aactgggtga atgtaataag tgatttgaaa aaaattgaag atcttattca atctatgcat 60

attgatgcta ctttatatac ggaaagtgat gttcacccca gttgcaaagt aacagcaatg 120

aagtgctttc tcttggagtt acaagttatt tcacttgagt ccggagatgc aagtattcat 180

gatacagtag aaaatctgat catcctagca caagatagtt tgtcttctaa tgggcaagta 240

acagaatctg gatgcaaaga atgtgaggaa ctggaggaaa aaaatattaa agaatttttg 300

cagagttttg tacatattgt ccaaatgttc atccaaactt ct 342

<210> 4

<211> 114

<212> PRT

<213> Artificial Synthesis ()

<400> 4

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ser

<210> 5

<211> 654

<212> DNA

<213> Artificial Synthesis ()

<400> 5

gcacctgagt tcgaaggggg accatcagtc ttcctgttcc ccccaaaacc caaggacact 60

ctcatgatct cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccaggaagac 120

cccgaggtcc agttcaactg gtacgtggat ggcgtggagg tgcataatgc caagacaaag 180

ccgcgggagg agcagttcca aagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 240

caggactggc tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg cctcccgtcc 300

tccatcgaga aaaccatctc caaagccaaa gggcagcccc gagagccaca ggtgtacacc 360

ctgcccccat cccaggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 420

ggcttctacc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 480

tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcagacta 540

accgtggaca agagcaggtg gcaggagggg aatgtcttct catgctccgt gatgcatgag 600

gctctgcaca accactacac acagaagagc ctctccctgt ctctgggtaa ataa 654

<210> 6

<211> 217

<212> PRT

<213> Artificial Synthesis ()

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Lys Ser Leu Ser Leu Ser Leu Gly Lys

210 215

<210> 7

<211> 45

<212> DNA

<213> Artificial Synthesis ()

<400> 7

ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttct 45

<210> 8

<211> 15

<212> PRT

<213> Artificial Synthesis ()

<400> 8

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

1 5 10 15

<210> 9

<211> 897

<212> DNA

<213> Artificial Synthesis ()

<400> 9

atcacgtgcc ctccccccat gtccgtggaa cacgcagaca tctgggtcaa gagctacagc 60

ttgtactcca gggagcggta catttgtaac tctggtttca agcgtaaagc cggcacgtcc 120

agcctgacgg agtgcgtgtt gaacaaggcc acgaatgtcg cccactggac aacccccagt 180

ctcaaatgca ttagagacgg tggtggtggt tctggtggtg gtggttctgg tggtggtggt 240

tctgcacctg agttcgaagg gggaccatca gtcttcctgt tccccccaaa acccaaggac 300

actctcatga tctcccggac ccctgaggtc acgtgcgtgg tggtggacgt gagccaggaa 360

gaccccgagg tccagttcaa ctggtacgtg gatggcgtgg aggtgcataa tgccaagaca 420

aagccgcggg aggagcagtt ccaaagcacg taccgtgtgg tcagcgtcct caccgtcctg 480

caccaggact ggctgaacgg caaggagtac aagtgcaagg tctccaacaa aggcctcccg 540

tcctccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagagcc acaggtgtac 600

accctgcccc catcccagga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 660

aaaggcttct accccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 720

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaga 780

ctaaccgtgg acaagagcag gtggcaggag gggaatgtct tctcatgctc cgtgatgcat 840

gaggctctgc acaaccacta cacacagaag agcctctccc tgtctctggg taaataa 897

<210> 10

<211> 298

<212> PRT

<213> Artificial Synthesis ()

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

290 295

Claims

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:

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.