CN114438075A

CN114438075A - Screening method and application of bacillus subtilis proprotease 9 targeted binding protein

Info

Publication number: CN114438075A
Application number: CN202210179591.8A
Authority: CN
Inventors: 刘博巽
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-05-06

Abstract

The invention discloses preparation and application of a subtilisin proprotein 9(PCSK9) targeted binding protein. The invention screens and prepares a PCSK9 targeted binding protein by a phage display peptide technology, and provides a convenient, efficient, stable and low-cost selection for the treatment and management of hypercholesterolemia and atherosclerotic cardiovascular diseases.

Description

Screening method and application of subtilisin 9 targeted binding protein

Technical Field

The invention belongs to the technical field of biological medicines, and relates to preparation of a subtilisin proprotein 9(PCSK9) targeted binding protein and application thereof in hypercholesterolemia and atherosclerotic cardiovascular disease (ASCVD).

Background

Hypercholesterolemia is an important pathophysiological basis for atherosclerotic disease. Large doses of statins are recommended in lipid lowering guidelines as a means of blood cholesterol management in high risk patients (e.g., ASCVD), but there are contradictions between therapeutic goals and adverse responses, including: the guidelines require serum LDL-cholesterol <1.8mmol/L in ASCVD patients, an extremely strict goal for patients with higher baseline LDL-cholesterol levels and monotherapy; (II) most of the large-dose statins have the risk of causing adverse reactions such as liver function damage, striated muscle damage and the like, and are difficult to tolerate by some patients; (iii) moderate doses of statin + cholesterol uptake inhibitor/bile acid excretion promoter have limited efficacy. Based on the above practical difficulties, therapeutic methods aiming at new targets are increasingly gaining attention. With the gradual confirmation that PCSK9 synthesized and secreted by hepatocytes plays a role in hyperlipidemia through degradation of LDLR, the PCSK9 monoclonal antibody (mAb) therapeutic drug provides a new important approach for the treatment and management of hypercholesterolemia and even ASCVD. However, mAb therapy has certain inherent drawbacks, including: whether humanized or not, the mAb contains an Fc segment, which may cross-link with immune cells and may even cause unintended immune responses; mAb has potential to induce anti-drug antibody (ADA) in vivo (both Epouluezumab and bococizumab are reported); and the ADA of bococizumab is one of the important reasons for marketing failure due to the fact that the ADA can generate cross resistance with other similar drugs; the preparation of the mAb depends on eukaryotic cells, the preparation process is complex, the cost is high, the clinical popularization of the medicines is insufficient, and the question of the drug effect/expense ratio of the mAb appears in the academia. Therefore, it is imperative to develop a convenient, efficient, stable, low-cost PCSK9 targeted binding protein that is beneficial for hypercholesterolemia and ASCVD management.

Disclosure of Invention

The invention aims to screen and prepare a PCSK9 targeted binding protein by using a phage display peptide technology, and perform functional verification on the PCSK9 targeted binding protein. The invention provides a convenient, efficient, stable and low-cost choice for the treatment and management of hypercholesterolemia and ASCVD.

The technical scheme adopted by the invention is as follows: a screening method of Bacillus subtilis proprotease 9 targeted binding protein is characterized in that: the subtilisin 9 is PCSK 9; the screening method comprises the following steps: screening a protein which is targeted and combined with PCSK9 from a phage display library by using a phage display technology, gene sequencing and a plasmid stable transformation large-scale expression technology, wherein the phage display peptide screening technology comprises the steps of incubating the phage display library in a culture dish coated with the PCSK9 protein, washing by using a phosphate buffer solution containing 0.5% of Tween, and obtaining the phosphate buffer solution containing 0.5% of Tween as PBST; eluting non-binding clones by using 0.5% PBST, fully amplifying the reserved binding clones after infecting escherichia coli, using VCSM13 to assist the precipitation after the phage rescue fermentation for extraction, and repeatedly washing after polyethylene glycol sedimentation for purification to obtain the binding phage collected after screening; the gene sequencing and plasmid stable transformation expression technology comprises the following steps: sequencing the DNA of the selected and collected combined phage, converting the nucleotide sequence obtained by sequencing into a protein amino acid sequence, comparing, preparing a proper sequence into a plasmid, and expressing and synthesizing by virtue of an escherichia coli system; the PCSK9 targeted binding protein is a protein with a linear structure, consists of 153 amino acid residues and does not contain a disulfide bond; can be specifically combined with PCSK9 protein synthesized and secreted by cells. The Bacillus subtilis proprotease 9 targeted binding protein and the application thereof are characterized in that: the PCSK9 targeted binding protein is applied to treatment of hypercholesterolemia and atherosclerotic cardiovascular diseases.

Compared with the prior art, the invention has the beneficial effects that: the target binding protein can be prepared through prokaryotic cells, is obviously different from mAb rich in disulfide bonds, and overcomes the storage difficulty caused by complex process for constructing hybridoma, high cost depending on a eukaryotic cell culture system and easy degradation at the temperature of 4 ℃ and above in the existing mAb preparation method; has the characteristics of simple and convenient process, low cost, easy storage and avoidance of repeated freeze thawing.

The targeted binding protein does not contain an Fc segment of the mAb, has a molecular weight far smaller than that of the mAb, and has lower possibility of generating cross-linking interaction with immune cells, generating non-targeted immune response and inducing ADA than the mAb, thereby avoiding the inherent problem of inducing non-targeted immune response by mAb drugs reported in clinical research.

Meanwhile, the targeted binding protein is easy to modify through various existing molecular biology technologies, including coupling of small molecule drugs, coupling of small molecule markers, coupling of self or other proteins, so that the targeted binding protein can be applied to multiple purposes of clinical and basic research.

Drawings

FIG. 1 is a schematic view; scheme for screening PCSK9 targeted binding protein by phage display technology

FIG. 2 is a schematic view; screening obtained PCSK9 specific dominant sequence

FIG. 3 is a schematic view; enzyme-linked immunosorbent assay (ELISA) method is adopted to verify the binding capacity of the target binding protein and PCSK9

FIG. 4 is a diagram of: validation of PCSK9 targeting binding protein function using HepG2, Huh7, et al human liver cell lines

Detailed Description

The invention is described in detail below with reference to the figures and specific embodiments.

As shown in fig. 1 to 4, the operation of the present invention is as follows:

screening of PCSK9 targeting binding protein in phage display library:

(1) adding 2ml of 100ug/ml recombinant human PCSK9 protein solution into a dish which is opened and placed and is irradiated by ultraviolet rays for 1h to coat the dish overnight; after coating, the dish contents were aspirated well and blocked with 0.5% Tween in 0.5% phosphate buffer (0.5% PBST) containing 0.5% Bovine Serum Albumin (BSA) for 1h at 37 ℃.

(2) After repeated washing with 2ml of 0.5% PBST for several times, 2ml of phage display peptide library was added to the dish and incubated at room temperature for 2h to complete the incubation process.

(3) Carefully sucking out the phage solution in the dish, repeatedly cleaning with 2ml of 0.5% PBST, performing plate beating for multiple times, adding 3ml of acidic eluent, placing in a decoloring shaking table, washing for 20min at 4 ℃, adding 500uL of alkaline neutralizing solution, shaking and uniformly mixing, and completing the elution process to obtain the phage solution before amplification after screening. Small amounts of phage fluid can be taken to determine titer.

(4) And adding the screened phage solution before amplification into the escherichia coli seed solution in the logarithmic growth phase, placing the escherichia coli seed solution on a shaking table at 37 ℃ and 220rpm for infection for 1h, coating the escherichia coli seed solution on a large agar culture plate for 37 ℃ overnight to form colonies, and scraping the colonies into 100ml of liquid 2YT culture medium again until the logarithmic growth phase is reached to finish the amplification process.

(5) Adding VCSM13 phage (10^9pfu/mL), standing at 37 ℃ for 30min for rescue fermentation, centrifuging to remove the supernatant, changing to a new 2YT culture medium to remove bacteria, centrifuging again to remove the culture medium, adding PEG according to the original volume of 1/5, shaking violently, after the phage is settled at 4 ℃ overnight, centrifuging again at 3000 + 10000rpm and 4 ℃ for 15-30min, and removing the supernatant to finish the extraction process.

(6) Repeating the centrifugation step to fully remove the water; then blowing PBS2ml to dissolve the precipitate, subpackaging in an EP tube, centrifuging for 5min at 10000-. The titer was measured at 200ul and the remaining 4 degrees were saved.

(7) The "post-amplification phage fluid" can be repeated as needed for 2-3 cycles.

Second, large scale synthesis of PCSK9 targeted binding proteins:

(1) infecting Escherichia coli with the bacteriophage liquid, coating on small agar plate, growing overnight, selecting 20-40 single colonies, adding into centrifugal tube containing liquid culture medium, amplifying, rescuing, fermenting, and removing bacteria.

(2) And (3) centrifuging the fermentation liquor at normal speed and high speed to remove thallus precipitates, and then transferring the liquid phase of each centrifugal tube into a new EP tube to obtain a plurality of monoclonal phage liquid.

(3) Depositing phage in the 'monoclonal phage body fluid' by using PEG, centrifuging at high speed to obtain solid-phase phage, purifying with iodide and absolute ethyl alcohol under the condition of keeping out of the sun, centrifuging, purifying again with 70% ethyl alcohol at 4 ℃, centrifuging to volatilize the ethyl alcohol, adding double distilled water for purging, and performing gene sequencing.

(4) The results of gene sequencing are translated into protein sequences using software, where proteins can be efficiently synthesized are referred to as "active sequences". The effective sequence is used for synthesizing plasmid transfection escherichia coli, after full growth and fermentation, thalli are cracked, and proteins are selected and extracted, so that the target binding protein can be obtained.

And thirdly, verifying the binding capacity of the target binding protein to PCSK9 by an enzyme-linked immunosorbent assay (ELISA):

(1) targeted binding protein targeted binding

proteins #

3, 7, 8, 11, 15, 16 encoding the following were synthesized using an e.coli expression system and negative control targeted binding protein #0 was expressed, purified using a nickel column, and endotoxin removed to <0.1EU/μ g using a casseri endotoxin removal kit:

targeted binding protein #3 (SEQ #3 with His Tag at N-terminal)

Targeted binding protein #7 (SEQ #7 with His Tag at N-terminus)

Targeted binding protein #8 (SEQ #8 with His Tag at N-terminus)

Targeted binding protein #11 (SEQ #11 with His Tag at N-terminus)

Targeted binding protein #15 (SEQ #15 with His Tag at N-terminus)

Targeted binding protein #16 (SEQ #16 with His Tag at the N-terminus)

Targeted binding protein #0 (SEQ #0 with His Tag at N-terminus)

(2) The 96-well plate was coated with 2ml of endotoxin-removed recombinant human PCSK9 protein solution, followed by addition of phosphate buffer (0.5% PBST) containing 0.5% tween, washing, and blocking with blocking solution.

(3) After blocking, the cells were incubated with targeting binding

proteins #

3, 7, 8, 11, 15, 16, and 0 for 2h, each set of targeting binding proteins was washed off with PBS (0.5% PBST) containing 0.5% tween 20, and then diluted anti-His Tag secondary antibody was added, followed by incubation for 1h and washing with 0.5% PBST. Then adding TMB working solution into each hole under the environment of room temperature and light shielding, and after reacting for 45-90 seconds, changing the solution from colorless to blue. The reaction was stopped by adding 1mmol/L dilute hydrochloric acid, and the solution turned yellow from blue.

(4) Absorbance was measured as OD450 using a microplate reader.

And fourthly, functional verification of the targeted binding protein in a human liver cell line:

(1) distributing HepG2 and Huh7 cells in a 24-well plate according to 5x10^3 cells per well, completely culturing for 24 hours in a culture medium to ensure that the cells are attached to the wall, and then starving the cells with a serum-free culture medium for overnight;

(2) the first column was added to complete medium, the second column was added to complete medium containing wild-type PCSK9 protein, the third column was added to complete medium containing targeted binding protein, and the fourth column was added to complete medium containing PCSK9 protein and targeted binding protein. After incubation for 24h in an incubator at 37 ℃.

(3) After removing the original medium, DiI-conjugated low-density lipoprotein (DiI-LDL) was added and incubated at 37 ℃ in an incubator without light for 4 hours. After washing with PBS for three times, direct fluorescence microscopy was performed, and DiI-LDL which was visually absorbed by rhodamine channel appeared as red light.

(4) Adding 4% paraformaldehyde for fixation, sealing with sealing liquid, adding rabbit-derived primary anti-LDLR, and incubating overnight; after washing with PBS, alexa flourur 488-conjugated anti-rabbit IgG secondary antibody was added and incubated for 1 hour; after PBS cleaning, DAPI is added, fluorescence microscopy is carried out, a FITC channel is used for seeing that the cell membrane LDLR is green light, and the positive signal mode is obviously different from the red light signal.

In conclusion, this example provides how to screen and prepare a protein targeted to bind to PCSK9 by using phage display peptide technology, and the experimental results of functional identification of the protein.

The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the scope of the present invention in any way, and all technical solutions obtained by using equivalent substitution methods fall within the scope of the present invention.

The parts not involved in the present invention are the same as or can be implemented using the prior art.

Claims

1. A screening method of Bacillus subtilis proprotease 9 targeted binding protein is characterized in that: the subtilisin 9 is PCSK 9;

the screening method comprises the following steps: a targeted binding protein capable of being specifically bound with PCSK9 is screened from a phage display library by a phage display technology, a gene sequencing technology and a plasmid stable transformation large-scale expression technology.

2. The method of screening for subtilisin 9-targeted binding proteins of claim 1, wherein: the phage display peptide screening technology is characterized in that: after the phage display library is incubated in a culture dish coated with PCSK9 protein, washing the phage display library by using a phosphate buffer solution containing 0.5% Tween, wherein the phosphate buffer solution containing 0.5% Tween is 0.5% PBST; eluting non-binding clone with 0.5% PBST, infecting Escherichia coli with the retained binding clone, amplifying, fermenting with VCSM13 as helper phage, precipitating with polyethylene glycol, washing repeatedly, and purifying to obtain screened binding phage.

3. The method of screening for subtilisin 9-targeted binding proteins of claim 1, wherein: the gene sequencing and plasmid stable transformation expression technology comprises the following steps: and (3) sequencing the DNA of the selected and collected binding phage, converting a nucleotide sequence obtained by sequencing into a protein amino acid sequence, comparing the protein amino acid sequence, preparing a proper sequence into a plasmid, and expressing and synthesizing by virtue of an escherichia coli system.

4. The method of screening for subtilisin 9-targeted binding proteins of claim 1, wherein: the PCSK9 targeted binding protein is a protein with a linear structure, consists of 153 amino acid residues and does not contain a disulfide bond; can be specifically combined with PCSK9 protein synthesized and secreted by cells.

5. The subtilisin 9 targeted binding protein of any of claims 1 to 4, for use, characterized in that: the PCSK9 targeted binding protein is applied to treatment of hypercholesterolemia and atherosclerotic cardiovascular diseases.