NKG2D receptor protein easy to directionally couple and immunoadsorbent of NKG2D receptor protein

Abstract

The invention discloses an NKG2D receptor protein easy to directionally couple and an immunoadsorbent of the NKG2D receptor protein. The structure of the NKG2D receptor protein is Km-Fc-NKG2D or Km-NKG2D-his. The immunoadsorbent is synthesized by taking the NKG2D receptor protein as a ligand, and tumor-related diseases are treated in an immunoadsorption manner. 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%, the immunoadsorption is to remove excessive pathogenic components in blood (plasma) in a physical manner, the interference to normal components of an organism is small, and the synthesized adsorbent has broad spectrum; and the NKG2D ligand form existing in an exosome form is effectively adsorbed by controlling the pore size on a porous carrier. By screening the carrier, a Sephacryl S-1000 SF glucan carrier is selected, the pore size is large, the separation range is 5*105-1*108D, small particle molecules with the molecular weight less than 300-400 nm can be separated, and MIC molecules existing in the exosome form, such as MICA *008 ligand molecules, can be more effectively adsorbed.

Description

technical field

[0001] The invention belongs to the field of blood purification, and mainly relates to an immunoadsorbent for removing free NKG2D ligands in blood, in particular to an NKG2D receptor protein and an immunoadsorbent that is easy for directional coupling. Background technique

[0002] NKG2D is an activating receptor of NK (natural killer) cells. NKG2D is expressed in almost all NK cells and CD8 + T cells, also expressed in natural killer T cells (natural killer T cells, NKT), αβT cells, γδT cells, CD4 + A subset of T cells. NKG2D is the only receptor that can recognize MICA / B found in vivo so far, and it can cause target cell decomposition by activating effector cells (Li Feng, Research progress on the correlation between MICA / B molecules and diseases and its clinical application, 2016 ). NKG2D ligands include six cytomegalovirus glycoprotein UL16 (ULBP 1-6) binding proteins and MICA / B and the retinoic acid early transcription family (RAET1E, RAET1G, and RAE...

Examples

Embodiment 1

[0058] Example 1 NKG2D vector construction and transient transfection

[0059] Select pcDNA3.1 as the eukaryotic cell expression vector, the amino acid sequence of K5-Fc-NKG2D protein is shown in SEQ ID NO.: 1, the DNA sequence is shown in SEQ ID NO.: 2, and EcoRI and XbaI 2 restriction sites are designed, and the N-terminal Add 5 lysines, denoted as K 5 -Fc-NKG2D. Then through primer design, with K 5 -Fc-NKG2D is the gene sequence for the template synthesis of K3-Fc-NKG2D and K1-Fc-NKG2D.

[0060] The amino acid sequence of NKG2D-his protein is shown in SEQ ID NO.: 3, and the DNA sequence is shown in SEQ ID NO.: 4. EcoRI and XbaI 2 enzyme cutting sites are designed, with 6 his tags at the C-terminal, K 5- NKG2D-his protein, that is, the N-terminus of NKG2D-his protein is coupled with 5 consecutive lysines. Outsource the whole gene synthesis, then use Lipofectamine 2000 as the transfection reagent, the amount of DNA is 200ng / well, use Opti-MEM medium to prepare the transfe...

Embodiment 2

[0061] Example 2 Purification and WB identification of protein

[0062] Purification of Fc-NKG2D protein: take 1mL of protein A affinity chromatography filler, equilibrate 5 times column volume with PBS, filter the collected cell culture medium and load it to the adsorption column, then equilibrate 5 times column volume with PBS, and finally Elution was performed with pH=2.8, 100 mM glycine-HCl buffer, and neutralization was performed with Tris-HCl buffer, pH=8.8.

[0063] Purification of NKG2D-his protein: Take 1 mL of nickel affinity chromatography filler, equilibrate 5 times the column volume with PBS, filter the collected cell culture medium and load it on the adsorption column, and then equilibrate 5 times with PBS (containing 20mM imidazole) Column volume, and finally the collected protein was eluted with PBS solution containing 300mM imidazole.

[0064] Identification of protein: use 12% separation gel electrophoresis, block with 1% BSA after transfer, then add anti-NK...

Embodiment 3

[0066] Example 3 Effects of Different Carriers and Different Protein Designs on the Performance of Immunoadsorbent Fillers

[0067] Preparation of NKG2D immunosorbent:

[0068] 1) according to embodiment 1 and 2 expression purification collection protein;

[0069] 2) Take 5mL of Sephacryl S-1000SF microspheres and Sepharose 6FF microspheres, add 15mL of 0.01M sodium hydroxide, add 3.0mL of epichlorohydrin solution, 38°C, 120rpm, react for 2 hours, clean the packing, and drain;

[0070] 3) Then add 5mL of PBS solution, add 3.0mL of ethylenediamine solution, 38°C, 120rpm, react for 3h, wash the packing, and drain;

[0071] 4) Add 5mL of PBS solution, add 7.5mL of glutaraldehyde solution, 38°C, 120rpm, react for 3h, wash the filler, and drain;

[0072] 5) According to the feeding amount of 15mg / ml, add K 5 -Fc-NKG2D, K 3 -Fc-NKG2D, K 1 -Fc-NKG2D protein solution, the buffer solution is carbonic acid buffer solution with pH=8.3, 30°C, 120rpm, after reacting for 3 hours, wash ...

technical field

[0001] The invention belongs to the field of blood purification, and mainly relates to an immunoadsorbent for removing free NKG2D ligands in blood, in particular to an NKG2D receptor protein and an immunoadsorbent that is easy for directional coupling. Background technique

[0002] NKG2D is an activating receptor of NK (natural killer) cells. NKG2D is expressed in almost all NK cells and CD8 + T cells, also expressed in natural killer T cells (natural killer T cells, NKT), αβT cells, γδT cells, CD4 + A subset of T cells. NKG2D is the only receptor that can recognize MICA / B found in vivo so far, and it can cause target cell decomposition by activating effector cells (Li Feng, Research progress on the correlation between MICA / B molecules and diseases and its clinical application, 2016 ). NKG2D ligands include six cytomegalovirus glycoprotein UL16 (ULBP 1-6) binding proteins and MICA / B and the retinoic acid early transcription family (RAET1E, RAET1G, and RAE...

Examples

Embodiment 1

[0058] Example 1 NKG2D vector construction and transient transfection

[0059] Select pcDNA3.1 as the eukaryotic cell expression vector, the amino acid sequence of K5-Fc-NKG2D protein is shown in SEQ ID NO.: 1, the DNA sequence is shown in SEQ ID NO.: 2, and EcoRI and XbaI 2 restriction sites are designed, and the N-terminal Add 5 lysines, denoted as K 5 -Fc-NKG2D. Then through primer design, with K 5 -Fc-NKG2D is the gene sequence for the template synthesis of K3-Fc-NKG2D and K1-Fc-NKG2D.

[0060] The amino acid sequence of NKG2D-his protein is shown in SEQ ID NO.: 3, and the DNA sequence is shown in SEQ ID NO.: 4. EcoRI and XbaI 2 enzyme cutting sites are designed, with 6 his tags at the C-terminal, K 5- NKG2D-his protein, that is, the N-terminus of NKG2D-his protein is coupled with 5 consecutive lysines. Outsource the whole gene synthesis, then use Lipofectamine 2000 as the transfection reagent, the amount of DNA is 200ng / well, use Opti-MEM medium to prepare the transfe...

Embodiment 2

[0061] Example 2 Purification and WB identification of protein

[0062] Purification of Fc-NKG2D protein: take 1mL of protein A affinity chromatography filler, equilibrate 5 times column volume with PBS, filter the collected cell culture medium and load it to the adsorption column, then equilibrate 5 times column volume with PBS, and finally Elution was performed with pH=2.8, 100 mM glycine-HCl buffer, and neutralization was performed with Tris-HCl buffer, pH=8.8.

[0063] Purification of NKG2D-his protein: Take 1 mL of nickel affinity chromatography filler, equilibrate 5 times the column volume with PBS, filter the collected cell culture medium and load it on the adsorption column, and then equilibrate 5 times with PBS (containing 20mM imidazole) Column volume, and finally the collected protein was eluted with PBS solution containing 300mM imidazole.

[0064] Identification of protein: use 12% separation gel electrophoresis, block with 1% BSA after transfer, then add anti-NK...

Embodiment 3

[0066] Example 3 Effects of Different Carriers and Different Protein Designs on the Performance of Immunoadsorbent Fillers

[0067] Preparation of NKG2D immunosorbent:

[0068] 1) according to embodiment 1 and 2 expression purification collection protein;

[0069] 2) Take 5mL of Sephacryl S-1000SF microspheres and Sepharose 6FF microspheres, add 15mL of 0.01M sodium hydroxide, add 3.0mL of epichlorohydrin solution, 38°C, 120rpm, react for 2 hours, clean the packing, and drain;

[0070] 3) Then add 5mL of PBS solution, add 3.0mL of ethylenediamine solution, 38°C, 120rpm, react for 3h, wash the packing, and drain;

[0071] 4) Add 5mL of PBS solution, add 7.5mL of glutaraldehyde solution, 38°C, 120rpm, react for 3h, wash the filler, and drain;

[0072] 5) According to the feeding amount of 15mg / ml, add K 5 -Fc-NKG2D, K 3 -Fc-NKG2D, K 1 -Fc-NKG2D protein solution, the buffer solution is carbonic acid buffer solution with pH=8.3, 30°C, 120rpm, after reacting for 3 hours, wash ...

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