CN113683710B

CN113683710B - NKG2D receptor protein easy to directionally couple and immunoadsorbent thereof

Info

Publication number: CN113683710B
Application number: CN202111094063.4A
Authority: CN
Inventors: 张海珍; 杨睿祯; 李家萍; 杨正根; 陈宇; 陈思锐; 陈校园
Original assignee: Guangzhou Kangsheng Biotechnology Co ltd
Current assignee: Guangzhou Kangsheng Biotechnology Co ltd
Priority date: 2021-09-17
Filing date: 2021-09-17
Publication date: 2023-09-12
Anticipated expiration: 2041-09-17
Also published as: CN113683710A

Abstract

The invention discloses NKG2D receptor protein easy to directionally couple and an immunoadsorbent thereof. The structure of the NKG2D receptor protein is Km-Fc-NKG2D or Km-NKG2D-his. According to the invention, the NKG2D receptor protein is taken as a ligand to synthesize the immunoadsorbent, and the immunoadsorption mode is used for treating the tumor-related diseases. The immunoadsorption can greatly reduce the content of soluble MIC molecules and ULBP molecules in a short time, the removal efficiency can reach 70% -97%, and the immunoadsorption is to remove the too high pathogenic components in blood (plasma) in a physical way, so that the interference on normal components of the organism is small, and the synthesized adsorbent has broad spectrum; by controlling the pore size on the porous support, NKG2D ligand forms present in exosome form are effectively adsorbed. By screening the carrier, sephacryl S-1000SF glucan carrier is selected, the pore diameter is larger, and the separation range is 5 x 10 ⁵ ‑1*10 ⁸ And D, small particle molecules with molecular weight less than 300-400nm can be separated, and MIC molecules existing in exosomes can be adsorbed more effectively, such as MICA 008 ligand molecules.

Description

NKG2D receptor protein easy to directionally couple and immunoadsorbent thereof

Technical Field

The invention belongs to the field of blood purification, and mainly relates to an immunoadsorbent for removing free NKG2D ligand in blood, in particular to NKG2D receptor protein easy for directional coupling and an immunoadsorbent thereof.

Background

NKG2D is a NK (natural killer) cell-activating receptor. NKG2D is expressed in almost all NK cells and CD8 ⁺ T cells, also expressed in natural killer T cells (natural killer T cell, NKT), alpha beta T cells, gamma delta T cells, CD4 ⁺ A subset of T cells. NKG2D is the only receptor found in vivo to date that can recognize MICA/B, causing target cell lysis by activating effector cells (Li Feng, research progress on the association of MICA/B molecules with disease and their clinical applications, 2016). NKG2D ligands include 6 cytomegalovirus glycoprotein UL16 (ULBP 1-6) binding proteins and MICA/B and retinoic acid early transcription family (RAET 1E, RAET1G, and RAET 1L). NKG2D can tolerate a high degree of variability in ligands using an induced-fit-mechanism (not shown) in recognizing different ligands. NKG2D has an elastic binding "pocket" that confers its ability to bind multiple ligands. Functionally, this ensures that NKG2D is able to recognize different ligands expressed on target cells under all stress or variant conditions, without being stimulated by any risk factors. Since NKG2D has a moderate binding force with its ligand, NKG2D must form a polymer to better bind to the ligand, so many published patents have modified NKG2D to enhance its activity. CN108367071a provides a NKG2D1-NKG2D2-Fc chimera for use in cancer immunotherapy, capable of enhancing ADCC. The patent states that dimeric NKG2D-Fc chimeras exhibit up to 100-fold improved binding avidity compared to the prior art monomeric NKG2D-Fc chimeras. WO2017083545A1 constructs multimers of 2-10 NKG2D extracellular domains via a C4b multimerization domain for the treatment of tumors or for the development of vaccines. AU2003296553A1 utilizes the characteristics that stress-induced endogenous dangerous signals HSP70 and MICA/B can synergistically activate NK cells to resist tumors, and develops corresponding pharmaceutical combinations for preventing and treating metastatic tumors. CN110636851A designed NKG2D extracellular domain-DAP 10-CD3 zeta chimeric receptor protein, developed treatment or prevention of nursingA medicament for cancer or infectious disease in a dairy animal.

MICA molecules can be classified into membrane MICA and soluble MICA (sMICA). Membrane type MICA/B high polymorphism, MICA has more than 100 polymorphisms, and MICB also has more than 33 recognizable alleleshttp:// hla.alleles.org/alleles/text_index.html). Wherein MICA site is highest in population and accounts for 10.1% -34.0%; MICA 002 accounts for 12.0% -21.7% of the population. The MICA 010 and MICA 019 duty ratios are also relatively high. MICB 005 sites were most frequently present in the population and were present at up to 57.4% in the population, among the allele distributions of MICB. The sources of secreted sMICA are mainly two, and one is that MICA of a membrane type is cut by metalloproteinase and then falls off; secondly, MICA 5.1 genes up-regulate expression of exosome secretion (VALLIAN S, RAD M J, et al Correlation of major histocompatibility complex class I related A (MICA) polymorphism with the risk of developing breast cancer, 2012). The cleavage reaction on the cell membrane is the main mechanism of NKG2DL production, such as sMICA, sMICB, sULBP production; sULBP3 is secreted by exosomes, which are isolated from pleural effusion of malignant mesothelioma and express TGFB and NKG2DL (MICA/B, ULPPs 1-3) on their surfaces, resulting in down-regulation of the NKG2D receptor by immune cells. Another investigator cultured Hela cells and found MICA 008 to exist mainly as full-length exosomes. Although MICA 019 and MICA 008 both down-regulate NKG2D expression and reduce cytotoxicity in CHO cells, MICA 008, which exists in exosome form, affects more (ashimu O B P, FERN NDEZ-mesina L, natural killer cell cytotoxicity is suppressed by exposure to the human NKG D ligand MICA 008that is shed by tumor cells in exosomes,2010). In terms of gene phenotype mica×010 is tightly linked to mica×a5, mica×008 is tightly linked to mica×a 5.1. The 5 exon allele MICA 5.1 of the transmembrane protein region encoding MICA molecule, with one G inserted in 5 GCTs, resulted in a frame shift mutation, leading to premature termination of the protein, resulting in the lack of truncated MICA molecules within the envelope. Of all MICA alleles currently found, only MICA x 008, MICA x 023, MICA x 028, MICA x 054 were closely linked to MICA x 5.1 (Li Pei, MICA, MICB genes)Polymorphism and acute myelogenous leukemia M2 type correlation study, 2015). In summary, the highest percentage of soluble sMIC proteins in the population were still sMICA 008, sMICA 002 and sMICB 005. Wherein sMICA x 008 exists in exosome form, sMICA x 002 and sMICB x 005 are cleaved and then are soluble in blood. The main effect of sMICA is to reduce CD8 ⁺ Expression of NKG2D by T cells and NK cells, thereby inhibiting the killing activity (Veronika Groh, tumour-derived soluble MIC ligands impair expression of NKG2D and T-cell activation, 2002). Therefore, after the sMICA in the blood is cleared, the targeting killing effect of NKG2D can be improved, and the treatment effect on tumors can be enhanced.

Immunoadsorption (IA) therapy is a blood purification technology developed in recent 15 years, and is to combine highly specific antigens, antibodies or substances (ligands) with specific physicochemical affinities with an adsorption material (carrier) to prepare an adsorbent (column), so as to selectively or specifically remove pathogenic factors in blood, thereby achieving the purposes of purifying blood and relieving illness. In recent years, immunoadsorption techniques such as protein a immunoadsorption and IgE immunoadsorption techniques have achieved good therapeutic effects in the treatment of autoimmune diseases. CN102988959a provides for the treatment and prevention of autoimmune diseases by modulating NKG2D to attenuate the expansion of autoreactive T cells or NK cells. The NKG2D is immobilized on a solid carrier to prepare an immunoadsorbent, and soluble NKG2D ligand molecules, including MICA, MICB, ULBP molecules and the like, can be removed efficiently in a blood purification mode, so that the aim of treating tumors by immunoadsorption is fulfilled.

The NKG2D receptor protein is generally coupled to a solid-phase carrier in a covalent coupling mode, and is mainly carried out by utilizing N or S atoms of the NKG2D receptor protein, so that the coupling efficiency is low, and the receptor protein is easy to fall off. Meanwhile, the coupling mode is difficult to control the coupling direction, and the improper coupling direction has a certain influence on the NKG2D receptor protein. The development of a better NKG2D immunoadsorbent preparation method is of great significance for improving the performance of the NKG2D immunoadsorbent.

Disclosure of Invention

The invention aims to overcome at least one defect of the prior art and provide NKG2D receptor protein which is easy to directionally couple and an immunoadsorbent thereof.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided:

an NKG2D receptor protein which is easy to directionally couple and has a structure of Km-Fc-NKG2D or Km-NKG2D-his, wherein: m=an integer from 3 to 5 (e.g. 3, 4 or 5).

In some examples of NKG2D receptor proteins, the amino acid sequence of Fc-NKG2D is as set forth in SEQ ID No.:1 is shown in the specification; the NKG2D amino acid sequence in said NKG2D-his is as set forth in SEQ ID No.: 3.

In some examples of NKG2D receptor proteins, the nucleotide sequence of Fc-NKG2D is as set forth in SEQ ID No.:2 is shown in the figure; the nucleotide sequence of the NKG2D-his is shown in SEQ ID NO.: 4.

In a second aspect of the invention, there is provided:

an expression vector capable of expressing the NKG2D receptor protein according to the first aspect of the present invention.

In some examples of expression vectors, the host cell is a eukaryotic cell or an insect cell.

In some examples of expression vectors, the host cells are CHO cells or 293ft cells.

In a third aspect of the invention, there is provided:

an NKG2D immunoadsorbent, which is obtained by covalently coupling an amino group of an NKG2D receptor protein according to the first aspect of the present invention to a solid carrier.

The solid phase carrier can be a solid phase carrier commonly used in the field, and has no special requirement. In some examples of NKG2D immunoadsorbers, the solid support is selected from at least one of chitosan, agarose, cellulose, dextran, resin.

In some examples of NKG 2D-immunoadsorbers, the solid support microspheres have a pore size in the range of 100-1200nm (e.g., 200nm, 300nm, 400nm, 500nm, 600nm, 700nm, 800nm, 900nm, 1000nm, 1100nm, etc.), and a separation of the globulins in the range of 10 ⁵ -10 ⁸ Da (e.g. 10 ⁶ Da level, 10 ⁷ Da level, etc.). This allows more targeted removal of the protein of interest.

In some examples of NKG2D immunoadsorbers, the solid support microspheres have a pore size in the range of 200-800nm and a separation of 10 for globulin ⁷ -10 ⁸ Da。

In some examples of NKG2D immunoadsorbers, the solid support is Sephacryl S-1000SF dextran.

In some examples of NKG2D immunoadsorbers, the method of covalent coupling is epichlorohydrin or glycerol ether.

In some examples of NKG2D immunoadsorbers, NKG2D immunoadsorber synthesis includes the following operations:

s1) taking solid-phase carrier filler, adding 0.01M NaOH with 3 times of volume according to the carrier: the volume ratio of the epoxy chloropropane is 1 (0.3-0.9), the epoxy chloropropane solution is added, the temperature is 35-42 ℃, the rpm is 100-150, the filling is cleaned after the reaction is carried out for 1-4 hours, and the filling is pumped out;

s2) adding an equal volume of PBS solution into the filler pumped out in the step S1), and according to the carrier: the volume ratio of the ethylenediamine solution is 1 (0.3-0.9), the ethylenediamine solution is added, the temperature is 35-42 ℃, the rpm is 100-150, the filling is cleaned after the reaction is carried out for 1-5 hours, and the filling is pumped out;

s3) adding an equal volume of PBS solution into the filler after the step S2) is pumped, adding glutaraldehyde solution according to the volume ratio of 1 (1-2), reacting at 35-42 ℃ at 100-150 rpm for 1-5 h, cleaning the filler by reverse osmosis water, and carrying out suction filtration;

s4) adding Fc-NKG2D protein or NKG2D-his protein solution into the filler after the suction filtration in the step S3), wherein the buffer solution is carbonate buffer solution with the same volume as the filler, the pH value is 8.3, the temperature is 25-35 ℃, the rpm is 100-150, the filler is cleaned after the reaction is carried out for 1-5 hours, and the NKG2D immunoadsorbent is obtained after the suction filtration.

In some examples of NKG2D immunoadsorbers, the immunoadsorbers adsorb MIC molecules present in exosomes by way of pore adsorption.

In some examples of NKG2D immunoadsorbers, the MIC molecule present in exosome form is MICA 008.

In some examples of NKG2D immunoadsorbers, the immunoadsorbers may adsorb free soluble NKG2D ligand molecules in blood.

In some examples of solid NKG2D immunoadsorbers, the soluble NKG2D ligand molecule includes at least one of MICA 002, MICA 004, MICA 007, MICA 009, MICA 010, MICA 012, MICA 019, MICA 027, MICA 045, MICA 002, MICA 003, MICA 004, MICA 005, MICA 008, MICA 014, MICA 024, and MICB 026 molecules.

In a fourth aspect of the invention, there is provided:

the use of an NKG2D immunoadsorbent according to the third aspect of the present invention for the preparation of a blood purification agent.

In some examples of applications, the blood purifying agent comprises a blood purifying agent that adsorbs NKG2D ligands in human blood or plasma.

In some examples of applications, the NKG2D ligand includes soluble MICA, MICB molecules, ULBP molecules, and immune complexes of MICA/B and anti-MIC mab drugs; thereby relieving tumor immunosuppression caused by abnormal elevation of NKG2D ligand molecules.

In some examples of applications, the NKG2D immunoadsorbers adsorb MIC molecules present in exosomes by way of pore adsorption.

In some examples of application, the MIC molecule in exosome form is MICA x 008.

In some examples of applications, the NKG2D immunoadsorbers adsorb free soluble NKG2D ligand molecules in blood.

In some examples of use, the soluble NKG2D ligand molecule comprises at least one of MICA 002, MICA 004, MICA 007, MICA 009, MICA 010, MICA 012, MICA 019, MICA 027, MICA 045, MICB 002, MICB 003, MICB 004, MICB 005, MICB 008, MICB 014, MICB 024, and MICB 026 molecules.

In a fifth aspect of the invention, there is provided:

an adsorption column comprising an NKG2D immunoadsorber according to the third aspect of the invention.

In a sixth aspect of the invention, there is provided:

a blood adsorption device comprising an adsorption column according to the fifth aspect of the invention.

The beneficial effects of the invention are as follows:

in one aspect, the present invention synthesizes immunoadsorbers with NKG2D receptor protein as ligand and treats tumor related diseases in immunoadsorbed mode. The immunoadsorption can greatly reduce the content of soluble MIC molecules and ULBP molecules in a short time, the clearance efficiency can reach 70% -97%, and the immunoadsorption is to physically remove the too high pathogenic components in blood (plasma) so as to have less interference on normal components of an organism. In theory NKG2D is able to bind all MIC molecules as well as ULBP molecules, and synthetic adsorbents have a broad spectrum.

On the other hand, when synthesizing an adsorbent, the NKG2D ligand existing in the exosome form is effectively adsorbed by controlling the pore size on the porous carrier. By screening the carrier, sephacryl S-1000SF dextran carrier is selected, the pore diameter is larger, and the separation range is 5 x 10 ⁵ -1*10 ⁸ Small particle molecules with molecular weights less than 300-400nm can be isolated. MIC molecules in exosome form, such as MICA 008 ligand molecules, can be adsorbed more efficiently using it as a carrier.

Meanwhile, through sequence design, lysine is introduced into the N end, the lysine contains more than 2 amino acids and is primary amine, the reaction is easier, the lysine structure is simpler than arginine, asparagine and glutamine, and the influence on the activity of target protein after the addition is minimal. The Fc end of receptor protein Fc-NKG2D is coupled to an agarose carrier by introducing lysine, so that the active site of the NKG2D is exposed to the outside to the maximum extent, and the adsorption performance of the synthesized adsorbent to MIC proteins is improved.

The NKG2D receptor protein of some examples of the present invention, because the ends have a specific type and number of amino acid sequences, it is easier for the solid phase carrier to undergo a coupling reaction, and thus, the NKG2D receptor protein may be better coupled than the solid phase carrier not coupled to the amino acid sequences, thereby exposing the NKG2D binding site to the outside, and thus, the NKG2D immunoadsorbers obtained by coupling may better adsorb free soluble NKG2D ligand molecules in blood, such as MICA 002, MICA 004, MICA 007, MICA 009, MICA 010, MICA 012, MICA 019, MICA 027, MICA 045, MICA 002, MICA 003, MICA 004, MICA 005, MICA 008, MICA 014, MICA 024, and MICA 026 molecules.

According to the NKG2D receptor protein of some examples, 3-5 lysines are introduced into the N end, so that the Fc end of more receptor proteins Fc-NKG2D can be coupled to a solid-phase carrier, the active site of NKG2D is exposed to the outside to the maximum extent, and the adsorption performance of the synthesized adsorbent to MIC proteins is improved.

The NKG2D immunoadsorbers of some examples of the invention effectively adsorb NKG2D ligand forms present in exosome form by controlling pore size on the porous support.

The NKG2D immunoadsorbent of some examples of the invention uses Sephacryl S-1000SF glucan as a solid phase carrier, has larger pore diameter and 5-10 separation range ⁵ ～1*10 ⁸ Small particle molecules of 300-400nm can be separated, and MICA 008 ligand molecules can be adsorbed more effectively.

Drawings

FIG. 1 is a diagram showing the identification of Fc-NKG2D protein and NKG2D-his protein WB; m: marker (KD): 180, 130, 100, 70, 55, 40, 35, 25, 15, 10 are arranged from top to bottom in sequence; lane 1: FC-NKG2D protein; lane 2: NKG2D-his proteins;

FIG. 2 shows adsorption rates of Fc-NKG2D protein and NKG2D-his protein to various MICA;

FIG. 3 shows adsorption rates of Fc-NKG2D protein and NKG2D-his protein to various MICB.

Detailed Description

Previous studies by the inventors indicate that the extracellular domain of NKG2D may be unstable, so that both the extracellular domain alone (NKG 2D-his) and the addition of the Fc-terminus to increase the stability (Fc-NKG 2D) of the receptor protein expression and the performance of the coupled synthetic adsorbent were studied in the present invention. When a ligand is immobilized to a carrier, if the Fc terminus of the Fc-NKG2D molecule is exposed to the outside, the receptor protein NKG2D is linked to the carrier due to steric hindrance, resulting in failure to bind the ligand effectively, and the adsorption ability to MICA protein is lowered. According to the invention, through the design of the gene sequence, 3-5 lysines are added at the N end of the Fc-NKG2D sequence or the N end of the NKG2D-his sequence, the directional coupling of the Fc-NKG2D protein on a solid-phase carrier can be unexpectedly promoted, and the coupling efficiency is remarkably improved.

Some sMICA molecules exist in the form of exosomes with a diameter of 50-200nm, and some examples of the invention can efficiently remove the exosomes by strictly screening the pore size of the solid phase carrier, controlling the pore size to be 100-1200nm, preferably 200-800 nm.

The technical scheme of the invention is further described below by combining examples.

EXAMPLE 1 NKG2D vector construction and transient transfection

pcDNA3.1 is selected as eukaryotic cell expression vector, and the amino acid sequence of K5-Fc-NKG2D protein is shown in SEQ ID NO.:1, the dna sequence is set forth in SEQ ID No.:2, designing EcoRI and XbaI 2 enzyme cutting sites, adding 5 lysines at the N end, and marking as K ₅ -Fc-NKG2D. Then by primer design, K ₅ -Fc-NKG2D is used as a template to synthesize the gene sequence of K3-Fc-NKG2D and K1-Fc-NKG 2D.

The amino acid sequence of the NKG2D-his protein is set forth in SEQ ID No.:3, the dna sequence is set forth in SEQ ID No.:4, designing EcoRI and XbaI 2 enzyme cutting sites, carrying 6 his labels at the C end and K ₅ The N-terminus of the NKG2D-his protein, is coupled with 5 consecutive lysines. Total gene synthesis was performed outside the plant, then Lipofectamine 2000 was used as a transfection reagent, the amount of DNA was 200ng/well, the transfection complex was prepared and cells were incubated with Opti-MEM medium at the time of transfection, and after 30 hours of transfection, the cells were centrifuged to collect the cell culture supernatant.

Example 2 purification of proteins and WB identification

Purification of Fc-NKG2D protein: 1mL of protein A affinity chromatography packing is taken, 5 times of column volume is balanced by PBS, the collected cell culture solution is filtered and loaded on an adsorption column, 5 times of column volume is balanced by PBS, finally, elution is carried out by using a pH=2.8 and 100mM glycine-HCl buffer solution, and neutralization is carried out by using a Tris-HCl buffer solution with pH=8.8.

Purification of NKG2D-his protein: 1mL of nickel affinity chromatography packing is taken, 5 times of column volume is balanced by PBS, the collected cell culture solution is filtered and then is loaded on an adsorption column, 5 times of column volume is balanced by PBS (containing 20mM imidazole), and finally, the collected protein is eluted by PBS solution containing 300mM imidazole.

Identification of proteins: electrophoresis was performed with 12% separation gel, blocking with 1% BSA after transfer, then adding anti-NKG 2D primary antibody (abcam), adding goat anti-mouse secondary antibody, and developing with DAB developing solution. See fig. 1. The Fc-NKG2D molecular weight was about 55KD; the NKG2D-His molecular weight was about 35KD.

Fc-NKG2D、K ₁ -Fc-NKG2D、K ₃ -Fc-NKG2D, K6-Fc-NKG2D, K-Fc-NKG 2D and NKG2D-his, K1-NKG2D-his, K3-NKG2D-his, K5-NKG2D-his, K7-NKG2D-his proteins were prepared by the method of examples 1 and 2.

Example 3 Effect of different carriers and different protein designs on the Performance of immunoadsorption fillers

Preparation of NKG2D immunoadsorbers:

1) Purified collection proteins were expressed as in examples 1 and 2;

2) Taking 5mL of Sephacryl S-1000SF microspheres and Sepharose 6FF microspheres, adding 15mL of 0.01M sodium hydroxide, adding 3.0mL of epichlorohydrin solution, reacting at 38 ℃ and 120rpm for 2 hours, cleaning the filler, and pumping out;

3) Then adding 5mL of PBS solution, adding 3.0mL of ethylenediamine solution, reacting at 38 ℃ and 120rpm for 3 hours, cleaning the filler, and pumping out;

4) Then adding 5mL of PBS solution, adding 7.5mL of glutaraldehyde solution, reacting at 38 ℃ and 120rpm for 3 hours, cleaning the filler, and pumping out;

5) K is added according to the feeding amount of 15mg/ml ₅ -Fc-NKG2D、K ₃ -Fc-NKG2D、K ₁ -Fc-NKG2D protein solution, buffer is carbonic acid buffer with ph=8.3, 30 ℃,120rpm, washing the filler after 3h reaction, and pumping to obtain NKG2D immunoadsorbent;

6) Finally, the mixture is preserved by 20 percent ethanol.

MIC protein adsorption experiments

The above-mentioned synthetic filler and 2mL of recombinant MICA protein solution with concentration of 45. Mu.g/mL or 2mL of MICB protein solution with concentration of 25. Mu.g/mL (self-made) are respectively filled into 10mL of EP tube, and placed on a decolorizing shaker, and after the above-mentioned synthetic filler and 2mL of recombinant MICA protein solution are contacted for 1-2 h at room temperature and 100rpm, the filler is placed into a disposable affinity chromatography column, and the filler is drained.

The MICA and MICB contents in the supernatants before and after adsorption were measured by the micro BCA method, respectively, and the clearance was calculated. The solid phase carrier without protein was used as a blank. The experimental results are shown in table 1.

Table 1 comparison of clearance of MICA 008 by different solid supports

Type of solid support	Ligands	MICA 008 clearance (%)
			Sepharose 6FF	Fc-NKG2D	50.5
Sephacryl S-1000SF	Fc-NKG2D	65.7
			Sepharose 6FF	NKG2D-his	49.5
Sephacryl S-1000SF	NKG2D-his	66.0

Adsorption to MICA 008 is most critical for all MICA species. As can be seen from the above table, sephacryl S-1000SF has a significantly higher adsorption effect on MICA 008 than Sepharose 6FF because of the larger pore size. So Sephacryl S-1000SF was initially selected as the carrier for the synthetic immunoadsorbers.

TABLE 2 influence of lysine Length on adsorption Performance

MICA x 002 and MICB x 005 are also among the highest-content subtypes among all MIC proteins. As can be seen from the above table, the adsorption performance of the synthetic adsorbent filler to MICA x 002 and MICB x 005 increases greatly with increasing lysine length when lysine is added at the N-terminus. When the number of amino acids is increased to 5-7, the clearance rate is not increased any more, and the stable state is realized. Considering the influence on the protein structure and the influence of signal peptidase cleavage, 5 lysines, namely K5-Fc-NKG2D or K5-NKG2D-his structures, were finally selected.

Compared with Fc-NKG2D as ligand, the adsorption activity of Fc-NKG2D with 5 lysines coupled at the N end is greatly increased, the adsorption performance of MICA 002 is improved by 27.4%, and the adsorption performance of MICB 005 is improved by 28.2%; compared with NKG2D-his as ligand, the N-terminal coupled NKG2D-his with 5 lysines has increased adsorption activity, increased adsorption performance to MICA 002 by 15.8% and adsorption performance to MICB 005 by 10.6%. In a word, the N-terminal coupling of lysine can improve the direction of Fc-NKG2D coupling in the solid phase carrier, and the directional coupling is easy to realize.

Example 4: determination of adsorption Performance of synthetic adsorbents on respective MIC proteins

Expression of purified K according to examples 1 and 2 ₅ -Fc-NKG2D and K ₅ -NKG2D-his protein. The filler was synthesized as in example 3 using Sephacryl S-1000SF microspheres and the adsorption properties were determined. The results are shown in FIGS. 2 and 3. The clearance rate of the Fc-NKG2D adsorbent to various MICA proteins ranges from 70% to 97%, and the clearance rate of the NKG2D-his to various MICA proteins ranges from 59% to 73%. The clearance rate of Fc-NKG2D to several MICB proteins is 70% -86%. NKG2D-his clearance of several MICB proteins was about 60% -73%. The adsorption effect of Fc-NKG2D is obviously higher than that of NKG2D-his, and the Fc is probably closer to the natural conformation and the activity is higher.

Fc-NKG2D amino acid sequence

In the above amino acid sequence, the underlined part is a signal peptide, the bold type is a K5 sequence, the italic type is an Fc sequence, followed by a linker sequence (italic+bold type), and the last part is an NKG2D sequence.

Fc-NKG2D gene sequence

In the nucleotide sequence, an italic bold marked part is an enzyme cutting site, an underlined dotted line marked part is a signal peptide sequence and a lysine sequence, an underlined solid line marked part is a gene sequence of an Fc fragment, a rear bold part is a linker sequence, and a last small letter part is an NKG2D gene sequence and a terminator.

NKG2D-his amino acid sequence

IWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSK EDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV- HHHHHH(SEQ ID NO.：3)

In the above amino acid sequence, the underlined dotted line indicates the signal peptide and the K5 sequence, the underlined solid line indicates the NKG2D sequence, and the his tag sequence is at the end.

NKG2D-his gene sequence

In the nucleotide sequence, the italic bold marked part is an enzyme cutting site, the underlined dotted line marked part is a signal peptide sequence and a K5 sequence, the underlined solid line marked part is an NKG2D sequence, and the sequence is followed by a his tag sequence and a terminator.

The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.

<110> Guangzhou Kangsheng Biotech Co., ltd

<120> an NKG2D receptor protein easy for directional coupling and immunoadsorbent thereof

<130>

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 401

<212> PRT

<213> artificial sequence

<400> 1

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Lys Lys Lys Lys Lys Thr His Thr Cys Pro Pro Cys

20 25 30

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

35 40 45

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

50 55 60

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly

245 250 255

Ser Ile Trp Ser Ala Val Phe Leu Asn Ser Leu Phe Asn Gln Glu Val

260 265 270

Gln Ile Pro Leu Thr Glu Ser Tyr Cys Gly Pro Cys Pro Lys Asn Trp

275 280 285

Ile Cys Tyr Lys Asn Asn Cys Tyr Gln Phe Phe Asp Glu Ser Lys Asn

290 295 300

Trp Tyr Glu Ser Gln Ala Ser Cys Met Ser Gln Asn Ala Ser Leu Leu

305 310 315 320

Lys Val Tyr Ser Lys Glu Asp Gln Asp Leu Leu Lys Leu Val Lys Ser

325 330 335

Tyr His Trp Met Gly Leu Val His Ile Pro Thr Asn Gly Ser Trp Gln

340 345 350

Trp Glu Asp Gly Ser Ile Leu Ser Pro Asn Leu Leu Thr Ile Ile Glu

355 360 365

Met Gln Lys Gly Asp Cys Ala Leu Tyr Ala Ser Ser Phe Lys Gly Tyr

370 375 380

Ile Glu Asn Cys Ser Thr Pro Asn Thr Tyr Ile Cys Met Gln Arg Thr

385 390 395 400

Val

<210> 2

<211> 1218

<212> DNA

<213> artificial sequence

<400> 2

gaattcatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 60

actggcaaaa aaaaaaaaaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 120

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 180

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 240

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 300

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 360

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 420

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 480

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 540

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 600

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 660

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 720

tacacgcaga agagcctctc cctgtctccc ggcggcggcg gcagcggcgg cggcagcata 780

tggagtgctg tattcctaaa ctcattattc aaccaagaag ttcaaattcc cttgaccgaa 840

agttactgtg gcccatgtcc taaaaactgg atatgttaca aaaataactg ctaccaattt 900

tttgatgaga gtaaaaactg gtatgagagc caggcttctt gtatgtctca aaatgccagc 960

cttctgaaag tatacagcaa agaggaccag gatttactta aactggtgaa gtcatatcat 1020

tggatgggac tagtacacat tccaacaaat ggatcttggc agtgggaaga tggctccatt 1080

ctctcaccca acctactaac aataattgaa atgcagaagg gagactgtgc actctatgcc 1140

tcgagcttta aaggctatat agaaaactgt tcaactccaa atacgtacat ctgcatgcaa 1200

aggactgtgt gatctaga 1218

<210> 3

<211> 175

<212> PRT

<213> artificial sequence

<400> 3

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Lys Lys Lys Lys Lys Ile Trp Ser Ala Val Phe Leu

20 25 30

Asn Ser Leu Phe Asn Gln Glu Val Gln Ile Pro Leu Thr Glu Ser Tyr

35 40 45

Cys Gly Pro Cys Pro Lys Asn Trp Ile Cys Tyr Lys Asn Asn Cys Tyr

50 55 60

Gln Phe Phe Asp Glu Ser Lys Asn Trp Tyr Glu Ser Gln Ala Ser Cys

65 70 75 80

Met Ser Gln Asn Ala Ser Leu Leu Lys Val Tyr Ser Lys Glu Asp Gln

85 90 95

Asp Leu Leu Lys Leu Val Lys Ser Tyr His Trp Met Gly Leu Val His

100 105 110

Ile Pro Thr Asn Gly Ser Trp Gln Trp Glu Asp Gly Ser Ile Leu Ser

115 120 125

Pro Asn Leu Leu Thr Ile Ile Glu Met Gln Lys Gly Asp Cys Ala Leu

130 135 140

Tyr Ala Ser Ser Phe Lys Gly Tyr Ile Glu Asn Cys Ser Thr Pro Asn

145 150 155 160

Thr Tyr Ile Cys Met Gln Arg Thr Val His His His His His His

165 170 175

<210> 4

<211> 540

<212> DNA

<213> artificial sequence

<400> 4

gaattcatgg agacagacac actcctgcta tgggtactgc tgctctgggt tccaggttcc 60

actggcaaaa aaaaaaaaaa aatatggagt gctgtattcc taaactcatt attcaaccaa 120

gaagttcaaa ttcccttgac cgaaagttac tgtggcccat gtcctaaaaa ctggatatgt 180

tacaaaaata actgctacca attttttgat gagagtaaaa actggtatga gagccaggct 240

tcttgtatgt ctcaaaatgc cagccttctg aaagtataca gcaaagagga ccaggattta 300

cttaaactgg tgaagtcata tcattggatg ggactagtac acattccaac aaatggatct 360

tggcagtggg aagatggctc cattctctca cccaacctac taacaataat tgaaatgcag 420

aagggagact gtgcactcta tgcctcgagc tttaaaggct atatagaaaa ctgttcaact 480

ccaaatacgt acatctgcat gcaaaggact gtgcatcatc atcatcatca ttgatctaga 540

Claims

1. An NKG2D receptor protein that is readily directionally coupled, characterized in that: the structure is Km-Fc-NKG2D or Km-NKG2D-his, wherein: k is lysine residue, m is residue number, m=3-5 integer, and Fc has amino acid sequence ofTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPThe amino acid sequence of NKG2D isIWSAVFLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLL KLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQRTV。

2. The NKG2D receptor protein of claim 1, wherein: the amino acid sequence of the Fc-NKG2D is shown in SEQ ID NO.:1 is shown in the specification; the NKG2D amino acid sequence in said NKG2D-his is as set forth in SEQ ID No.:3 is shown in the figure;

the nucleotide sequence of the Fc-NKG2D is shown as SEQ ID NO.:2 is shown in the figure; the nucleotide sequence of the NKG2D-his is shown in SEQ ID NO.: 4.

3. An expression vector expressing the NKG2D receptor protein of claim 1 or 2.

4. The expression vector of claim 3, wherein: the host cell is eukaryotic or insect.

5. The expression vector of claim 3, wherein: the host cell is CHO cell or 293ft cell.

6. An NKG2D immunoadsorber obtained by covalently coupling the NKG2D receptor protein of claim 1 or 2 to a solid support via an amino group.

7. The NKG2D immunoadsorber of claim 6, wherein: the solid phase carrier is at least one selected from chitosan, agarose, cellulose, dextran and resin.

8. The NKG2D immunoadsorber of claim 7, wherein: the solid phase carrier is microsphere with pore diameter of 100-1200nm and separation range of globulin of 10 ⁵ -10 ⁸ Da。

9. The NKG2D immunoadsorber of claim 8, wherein: the aperture range of the microsphere is 200-800nm, and the separation range of the microsphere to globulin is 10 ⁷ -10 ⁸ Da。

10. The NKG2D immunoadsorber of claim 7, wherein: the solid phase carrier is Sephacryl S-1000SF glucan.

11. The NKG2D immunoadsorber according to any of claims 6 to 10, wherein: the covalent coupling method is an epichlorohydrin method or a glycerol ether method.

12. The NKG2D immunoadsorber of claim 11, wherein: NKG2D immunoadsorber synthesis involves the following operations:

s4) adding Km-Fc-NKG2D protein or Km-NKG2D-his protein solution into the filler after the suction filtration in the step S3), wherein the buffer solution is carbonate buffer solution with the same volume as the filler, the pH value is 8.3, the temperature is 25-35 ℃, the speed is 100-150 rpm, the filler is cleaned after the reaction is carried out for 1-5 hours, and the NKG2D immunoadsorbent is obtained after the suction filtration.

13. Use of an immunoadsorbent as claimed in any of claims 6 to 12 for the preparation of a blood purification agent.

14. The use according to claim 13, characterized in that: the blood purifying agent includes a blood purifying agent that adsorbs NKG2D ligands in human blood or plasma.

15. The use according to claim 14, characterized in that: the NKG2D ligand comprises soluble MICA, MICB molecule, ULBP molecule and immune complex composed of MICA/B and anti-MIC monoclonal antibody.

16. The use according to claim 14, characterized in that: the immunoadsorbent adsorbs MIC molecules existing in exosomes by means of pore adsorption.

17. The use according to claim 16, characterized in that: the MIC molecule in exosome form is MICA x 008.

18. The use according to claim 14, characterized in that: the immunoadsorbent can adsorb free soluble NKG2D ligand molecules in blood.

19. The use according to claim 18, characterized in that: the soluble NKG2D ligand molecule includes at least one of MICA 002, MICA 004, MICA 007, MICA 009, MICA 010, MICA 012, MICA 019, MICA 027, MICA 045, MICB 002, MICB 003, MICB 004, MICB 005, MICB 008, MICB 014, MICB 024, and MICB 026 molecules.

20. An adsorption column comprising the NKG2D immunoadsorber of any of claims 6-12.

21. A blood adsorption device comprising the adsorption column of claim 20.