CN109395616B

CN109395616B - Preparation method of nucleoporin grafted homogeneous pore polycarbonate bionic protein transport membrane

Info

Publication number: CN109395616B
Application number: CN201811501825.6A
Authority: CN
Inventors: 王明霞; 严峰; 曹越超; 李晓丹; 宋健; 王文昭
Original assignee: Tianjin Polytechnic University
Current assignee: Tianjin Polytechnic University
Priority date: 2018-12-10
Filing date: 2018-12-10
Publication date: 2021-03-09
Anticipated expiration: 2038-12-10
Also published as: CN109395616A

Abstract

The invention discloses a preparation method of a nucleoporin grafted homogeneous pore polycarbonate bionic protein transmission membrane. The method comprises the following steps: (1) hydroxylating the surface of the polycarbonate mesoporous film by adopting plasma irradiation or sulfide oxidation; (2) reacting the surface hydroxylated polycarbonate film with diisocyanate to convert surface terminal hydroxyl groups into isocyanate (-NCO) groups; (3) reacting the film treated by diisocyanate with diamine to aminate the surface of the film; (4) the surface aminated membrane is reacted with a bifunctional protein cross-linking agent 4- (p-maleimide phenyl) butyric acid succinimide ester to enable the surface of the membrane to have high-activity maleimide; (5) the Sulfhydryl (SH) at the tail end of nucleoporin (NSP1) is bonded with maleimide on the surface of the membrane to graft nucleoporin to the surface of the polycarbonate homogeneous pore membrane, so as to prepare the nucleoporin grafted homogeneous pore polycarbonate bionic membrane which can be used for selectively transmitting protein.

Description

Preparation method of nucleoporin grafted homogeneous pore polycarbonate bionic protein transport membrane

Technical Field

The invention relates to the field of biological separation, in particular to a preparation method of a nucleoporin grafted homogeneous pore polycarbonate bionic protein transmission membrane.

Background

Protein drugs are used for diagnosing, preventing and treating diseases at the protein level, and the protein drugs in the broad sense include all products with the chemical nature of protein or polypeptide, such as hormones, growth factors/cytokines, proteases, receptor molecules, monoclonal antibodies and antibody-related molecules, partial protein or polypeptide vaccines and the like. Compared with the existing small molecule drugs, the protein drug has the characteristics of high activity, strong specificity, low toxicity, clear biological function and contribution to clinical application. Because of its high success rate, safety and reliability, it has become an important component in medical products. However, naturally occurring proteins, or proteins produced by fermentation, culture or synthesis, always consist of a plurality of components in the initial stage, and have to be separated and purified for use in the fields of medicine, food, and the like. Therefore, the high-efficiency and selective separation research of the protein has important theoretical significance and practical application value, and becomes a hotspot of modern drug analysis, life science, biological engineering and chemical engineering research.

The means currently used for protein separation are mainly chromatography, including gel chromatography, ion exchange chromatography, high performance liquid chromatography, and the like. However, the equipment required by these techniques is expensive and has a small processing amount, and industrial production application is difficult to realize. In recent years, affinity chromatography is widely applied to separation and purification of biological macromolecules such as protein, nucleic acid, polypeptide and the like with good separation performance, and the method achieves the separation purpose based on the mutual recognition effect between ligand and target biological molecules, such as antibody and antigen, sugar and lectin, enzyme and substrate and the like, and is one of the most effective means in the current macromolecular separation mechanism, however, the operation of the method is easily limited by mass transfer and the treatment capacity is small.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a preparation method of a nucleoporin grafted homogeneous pore polycarbonate bionic protein transmission membrane.

The invention relates to a preparation method of a nucleoporin grafted homogeneous pore polycarbonate bionic protein transmission membrane, which comprises the following steps: (1) hydroxylating the surface of the polycarbonate mesoporous film by adopting plasma irradiation or sulfide oxidation; (2) reacting the surface hydroxylated polycarbonate film with diisocyanate to convert surface terminal hydroxyl into isocyanate-NCO group; (3) the film treated by diisocyanate reacts with alkalescent diamine to aminate the surface of the film; (4) the surface aminated membrane is reacted with a bifunctional protein cross-linking agent 4- (p-maleimide phenyl) butyric acid succinimide ester to enable the surface of the membrane to have high-activity maleimide; (5) bonding a Sulfhydryl (SH) at the tail end of nucleoporin (NSP1) with maleimide on the surface of the membrane to graft nucleoporin onto the surface of the polycarbonate homogeneous pore membrane to prepare a nucleoporin grafted homogeneous pore polycarbonate bionic membrane; wherein the plasma source for plasma irradiation is a mixture of hydrogen and helium, the volume ratio of the hydrogen to the helium is 0.5-2: 99, the flow is 10-40L/min, and the power is 120-220W; the persulfate is one or a mixture of ammonium persulfate, potassium persulfate and sodium persulfate in any proportion; the average pore diameter of the polycarbonate homogeneous pore membrane is 10 nm-2000 nm; the diisocyanate comprises one or a mixture of 1, 6-hexamethylene diisocyanate, toluene 2, 6-diisocyanate and 1, 5-naphthalene diisocyanate in any proportion; in order to avoid the dissolution of the polycarbonate film, weak alkaline diamine such as p-phenylenediamine or m-phenylenediamine is adopted to carry out surface amination treatment on the film. The nucleoporin grafted homogeneous pore polycarbonate bionic protein transmission membrane prepared by the method can realize the selective transportation of the nucleoplasm transporter Kaps 95.

Drawings

FIG. 1 is a schematic view of a protein separation apparatus used in example 1 of the present invention.

Detailed Description

The invention is further illustrated by the following examples, which are intended to better understand the content of the invention and not to limit the scope of protection of the invention:

example 1

(1) Oxidizing a polycarbonate mesoporous membrane by using potassium persulfate, preparing the potassium persulfate into an aqueous solution with the mass concentration of 20%, soaking the polycarbonate mesoporous membrane in the potassium persulfate solution, and reacting for 5 hours at 80 ℃ to obtain a polycarbonate membrane with the hydroxylated surface; (2) preparing acetonitrile solution of toluene 2, 6-diisocyanate with the mass fraction of 5%, soaking a polycarbonate film with hydroxylated surface in the toluene 2, 6-diisocyanate solution, and reacting at 60 ℃ for 10min to convert the hydroxyl at the terminal surface into isocyanate-NCO group; (3) preparing an acetonitrile solution of m-phenylenediamine with the mass fraction of 5%, soaking the film with the surface treated by diisocyanate in the m-phenylenediamine solution, and reacting at 60 ℃ for 10min to aminate the surface of the film; (4) dissolving a bifunctional protein cross-linking agent 4- (p-maleimide phenyl) butyric acid succinimide ester (SMPB) in a phosphate buffer solution with the pH value of 7.4, wherein the concentration of the SMPB is 2mmol/L, and then placing a polycarbonate membrane with active amino on the surface in the SMPB solution to react for 15min at room temperature to obtain a maleimide functionalized polycarbonate membrane; (5) dissolving a nucleoporin (NSP1) stock solution into a PBS buffer solution with the pH value of 7.4, wherein the concentration of NSP1 is 0.2mg/mL, and then soaking the maleimide functionalized polycarbonate membrane into an NSP1 solution for reaction for 20min to obtain the nucleoporin grafted homogeneous pore polycarbonate bionic membrane.

The nucleoporin modified polycarbonate membrane is fixed in the middle of a diffusion cell shown in figure 1, the left chamber and the right chamber are isolated, the right chamber is filled with a sample solution (stock solution, the protein concentration is 0.5mg/mL) of protein to be separated (nuclear plasma transfer protein Kaps95 or bovine serum albumin BSA), the left chamber is filled with a buffer solution, and the volumes of the two chambers are both 5 mL. According to the concentration principle, proteins will diffuse across the membrane from the right compartment to the left compartment. In order to enlarge the concentration difference between the two chambers, the left chamber is externally connected with a diluting pool (1000mL) with larger volume through a pipeline so as to increase the driving force of the concentration difference and enable the protein to diffuse to the left chamber more quickly. Samples were taken from the left and right chambers at different experimental times and the protein concentration was measured using a microplate reader. After diffusion for 24h, the concentration of left ventricular Kaps95 increased from 0mg/mL to 0.18mg/mL, while BSA hardly diffused to the left ventricle under the same conditions. The result shows that the nucleopore protein grafted homogeneous pore polycarbonate bionic membrane has selectivity on Kaps95 protein and is expected to be applied to the selective separation of protein.

Claims

1. A preparation method of a nucleoporin grafted homogeneous pore polycarbonate bionic protein transmission membrane is characterized by comprising the following steps: (1) hydroxylating the surface of the polycarbonate mesoporous film by adopting plasma irradiation or sulfide oxidation; (2) reacting the surface hydroxylated polycarbonate film with diisocyanate to convert surface terminal hydroxyl into isocyanate-NCO group; (3) the film treated by diisocyanate reacts with alkalescent diamine to aminate the surface of the film; (4) the surface aminated membrane is reacted with a bifunctional protein cross-linking agent 4- (p-maleimide phenyl) butyric acid succinimide ester to enable the surface of the membrane to have high-activity maleimide; (5) bonding a Sulfhydryl (SH) at the tail end of nucleoporin (NSP1) with maleimide on the surface of the membrane to graft nucleoporin onto the surface of the polycarbonate homogeneous pore membrane to prepare a nucleoporin grafted homogeneous pore polycarbonate bionic membrane;

the plasma source for plasma irradiation is a mixture of hydrogen and helium, the volume ratio of the hydrogen to the helium is 0.5-2: 99, the flow is 10-40L/min, and the power is 120-220W;

the persulfate is one or a mixture of ammonium persulfate, potassium persulfate and sodium persulfate in any proportion;

the average pore diameter range of the polycarbonate mesoporous membrane is 10 nm-2000 nm;

the diisocyanate comprises one or a mixture of 1, 6-hexamethylene diisocyanate, toluene 2, 6-diisocyanate and 1, 5-naphthalene diisocyanate in any proportion;

the weak alkaline diamine is p-phenylenediamine or m-phenylenediamine.

2. The method for preparing a nucleoporin grafted isoporous polycarbonate biomimetic protein transfer membrane according to claim 1, characterized in that the selective transportation of nuclear plasma transport protein Kaps95 can be realized.