CN116063381A

CN116063381A - Polypeptide targeting gp140 protein and application thereof

Info

Publication number: CN116063381A
Application number: CN202211399145.4A
Authority: CN
Inventors: 毛传斌; 岳慧; 杨明英
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-11-09
Filing date: 2022-11-09
Publication date: 2023-05-05

Abstract

The invention discloses a polypeptide targeting gp140 protein and application thereof. The amino acid sequence of the polypeptide is MHLHRTP, WPHHRHH, WPFHHKH respectively. The polypeptide can efficiently target gp140 envelope protein trimer, blocks gp140 envelope protein on HIV virus from being combined with CD4 receptor protein on immune cells, blocks the immune cells from being infected, further prevents the immune cells from being destroyed, inhibits HIV virus replication, has specific targeting binding capacity on gp140 protein, and provides a new thought for the development of clinical HIV antiviral drugs.

Description

Polypeptide targeting gp140 protein and application thereof

Technical Field

The invention belongs to a polypeptide in the biomedical field, and particularly relates to a novel polypeptide capable of being targeted to bind human immunodeficiency virus 1 (HIV-1) membrane protein gp140 and application thereof.

Background

Phage display technology is a technology in which a DNA sequence encoding an exogenous polypeptide or protein is inserted into a phage gene by genetic engineering, so that the polypeptide or protein is displayed on the surface of phage capsid protein. A large number of phages displaying different random polypeptides can constitute a phage library for screening against specific targets to explore the interactions between the polypeptides and the targets.

In the field of biology, this technique is widely used in the study of interactions between proteins, proteins and polypeptides, proteins and DNA, and the like.

Disclosure of Invention

In order to solve the problems existing in the background art, the invention aims to provide a polypeptide sequence which has high affinity with gp140 protein and can specifically target and bind gp140 protein, can be compatible with gp140 protein consisting of HIV surface membrane protein gp120 and transmembrane protein gp41 extracellular domain, and clarifies the application of the polypeptide in the HIV antiviral treatment field.

According to the invention, the gp140 targeting peptide is obtained through phage mass screening, and can specifically bind gp140 protein to block interaction between CD4 on the surface of immune cells and gp140 on the surface of HIV, thereby inhibiting HIV replication and destroying immune cells.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

1. A polypeptide targeting gp140 protein, wherein the amino acid sequence of the polypeptide is one of the amino acid sequences shown in No. 1-No. 3.

The following table:

TABLE 1 amino acid sequence listing

	Amino acid sequence
		No.1	MHLHRTP
No.2	WPHHRHH
		No.3	WPFHHKH

2. A bioactive substance comprising a polypeptide, and further comprising a covalently linked compound or nanoparticle, an engineered phage, or other microorganism, multimeric mixture.

The bioactive substance has the same biomedical function as the polypeptide, namely, the bioactive substance can be targeted to bind to gp140 protein and block the binding of gp140 envelope protein and CD4 receptor protein.

3. A polynucleotide sequence capable of encoding any one of the amino acid sequences, or capable of encoding the biologically active substance.

4. A polypeptide medicine for treating diseases contains said polypeptide, and is used for targeting gp140 protein, preventing immune cells from being destroyed and inhibiting HIV virus replication.

The invention can be used for preparing biomedical materials and medicines for HIV antiviral treatment by targeting gp140 protein.

The polypeptide can efficiently target gp140 envelope protein trimer, blocks the gp140 envelope protein on HIV virus from being combined with CD4 receptor protein on immune cells, blocks the immune cells from being infected, further prevents the immune cells from being destroyed, inhibits HIV virus replication, has specific targeting binding capacity on gp140 protein, and provides a new thought for the development of clinical HIV antiviral drugs.

The polypeptide of the invention can block the specific binding between gp140 and a surface antigen cluster 4 receptor (CD 4), thereby blocking HIV from penetrating into susceptible immune cells, cutting off HIV replication and further playing a role in inhibiting HIV from damaging an immune system.

The beneficial effects of the invention are as follows:

the present inventors used phage 7 peptide library screening to obtain polypeptide sequences capable of specifically binding gp140 protein. In contrast to the usual inhibitors of HIV, the advantages of gp140 binding peptides are:

(1) The sequence is short and small, and the synthesis production cost is low;

(2) The polypeptide fragment has stable structure, and reduces the transportation and storage cost and difficulty;

(3) Administration may be performed in a variety of ways.

In the field of HIV antiviral treatment, the specific targeting binding capacity is provided, and a new thought is developed for the research and development of inhibitor drugs.

Drawings

FIG. 1 shows phage output from 2-3 rounds of repeated screening during 4 rounds of screening for gp140 protein using phage 7 peptide library in example 1.

FIG. 2 shows the 3 polypeptide sequences with high occurrence frequency and the corresponding frequency after analysis of the sequencing result by DNAStar software in example 2.

FIG. 3 is a schematic representation of binding sites for a mimetic polypeptide and protein using the CABS-dock website, a mimetic binding site for a MHLHRTP (MP) polypeptide, a WPHHHHH (WPH) polypeptide, a WPFHHKH (WPF) polypeptide, and a gp140 protein in example 2.

FIG. 4 is a graph showing the affinity of phage displaying 3 different polypeptide sequences to gp140 protein by ELISA in example 3.

Detailed Description

The present invention is further illustrated by the following examples, which are given solely by way of illustration and not limitation, and various modifications and alterations of the invention will become apparent to those skilled in the art and are deemed to be within the spirit and principles of the invention as defined herein.

Embodiments of the invention are as follows:

example 1

4 rounds of screening (2-4 rounds of repeated screening) were performed against gp140 protein-specific binding positive polypeptides using a phage 7 peptide library constructed by phage display technology.

1.1 resuscitating and culturing host bacterium E.coil ER2738

LB solid culture plates were prepared and placed in an incubator at 37℃for 30min. And (3) using a fungus picking rod to pick E.coil ER2738 fungus liquid, coating the fungus liquid on the surface of an LB plate in a Z shape, and culturing in a 37 ℃ incubator for 12 hours. To the shake tube, 5mL of liquid LB medium containing tetracycline was added, and the monoclonal colonies in the plates were picked up using a sterile tip, and the tip was placed in the medium. The culture tubes were placed in a shaker at 37℃and shaking culture at 220rpm until the bacteria were in mid-log growth.

1.2 determination of phage titers

LB/IPTG/Xgal plates were placed in an incubator at 37℃for 1 hour to preheat. The phage solution was subjected to gradient dilution, and 10. Mu.L of the diluted phage solution was added to 200. Mu.L of E.coil ER2738 bacterial liquid in the logarithmic growth phase. Standing for 15min. The infected phage were applied to the LB/IPTG/Xgal plate surface and spread evenly using a sterile coated rod. The coated plates were placed in an incubator at 37℃overnight. Phage blue spots on the plates were checked the following day and counted.

1.3 preparation of bacterial liquid

20mL of LB medium was placed in a 250mL sterile conical flask, and 1mL of host bacterium E.coil ER2738 in mid-log growth was added. The Erlenmeyer flask was placed in a shaking table at 37℃and shaking culture at 220rpm until the bacteria were in mid-log growth.

1.4 amplification and purification of phages

The neutralized phage eluate was added to 20mL of E.coil ER2738 bacterial solution in logarithmic growth phase. After standing at room temperature for 20min, the bacterial liquid is placed in a shaking table at 37 ℃ and cultured for 4.5 hours at 220 rpm. The bacterial liquid was then poured into a 50mL centrifuge tube and centrifuged at 12000g for 20min. The supernatant was poured into a centrifuge tube containing 4mL of PEG/NaCl solution, placed in a refrigerator at 4℃and allowed to settle overnight. The next day, the centrifuge tube 12000g was centrifuged for 20min, and the supernatant was discarded. 1mL of PBS solution was added to the centrifuge tube, the pellet was redissolved, and the resuspension was transferred to a 1.5mL EP tube, placed in a shaker at 37℃and shaken for 1 hour. The EP tube 12000g was then centrifuged for 10min, and the supernatant was transferred to an EP tube containing 200. Mu.L of PEG/NaCl solution. EP tubes were placed at 4℃and left to stand for 1 hour to settle phages. After that, the EP tube 12000g was centrifuged for 20min, and the supernatant was discarded, and 100. Mu.L of PBS was added to resuspend the pellet.

Round 1 screening

1.5 coating of Gp140 protein

mu.L of gp140 protein solution was added to a 24-well plate and the 24-well plate was placed in a wet box. The wet box was placed on a rocker and coated overnight at 4 ℃.

1.6 coating of Gp140 antibody

The liquid in the 24-well plate was blotted using a pipette and the plate was spun down on sterile filter paper to thoroughly remove the liquid. mu.L of gp140 antibody solution was added to the 24-well plate and the 24-well plate was placed in a wet box and coated overnight at 4 ℃.

Blocking of Gp140/Gp140 antibodies

The liquid in the 24-well plate was blotted using a pipette and the plate was spun down on sterile filter paper to thoroughly remove the liquid. 2mL of 0.5% BSA blocking solution was added. The well plate was placed on a rocker shaker and closed for 1 hour.

1.8 washing

The liquid in the 24-well plate was washed using a pipette and the plate was spun down on sterile filter paper to thoroughly remove the liquid. 1mL of sterile PBST solution was added and the well plate was placed on a rocker shaker. After 5min, the liquid in the well plate was again pipetted and beaten, 1mL of sterile PBST solution was added and the well plate was placed on a rocker shaker. This step was repeated 6 times to thoroughly wash out the excess blocking solution in the well plate.

1.9 binding of phage 7 peptide library to gp140 protein

Sterile EP tubes were taken and 1mL of PBS solution and 10. Mu.L (2X 10) ¹¹ pfu) 7 peptide phage library, were mixed by vortexing. The mixture was added to a 24-well plate and the plate was placed on a rocker shaker and incubated for 1 hour at room temperature.

1.10 collecting unbound phage

The liquid in the 24-well plate was collected into sterile EP tubes using a pipette. A small amount of sterile PBS solution was added for washing, ensuring that unbound phage were all collected.

Two to four wheel repeat screening

1.11 coating of Gp140 protein

1.12 blocking of the Gp140 protein

The liquid in the 24-well plate was washed using a pipette and the plate was spun down on sterile filter paper to thoroughly remove the liquid. 2mL of 0.5% BSA blocking solution was added. The well plate was placed on a rocker shaker and closed for 1 hour.

1.13 washing

1.14 binding of phage libraries to gp140 protein

Sterile EP tubes were taken and 1mL of PBS solution was added to phage sub-library (2X 10) ¹¹ pfu), by vortexing. The mixture was added to a 24-well plate and the plate was placed on a rocker shaker and incubated for 1 hour at room temperature.

1.15 washing

The liquid in the 24-well plate was washed using a pipette and the plate was spun down on sterile filter paper to thoroughly remove the liquid. 1mL of sterile PBST solution was added and the well plate was placed on a rocker shaker. After 5min, the liquid in the well plate was again pipetted and beaten, 1mL of sterile PBST solution was added and the well plate was placed on a rocker shaker. This step was repeated 10 times to thoroughly wash phage in the well plate that did not bind or did not bind firmly to gp140 protein.

1.16 elution and neutralization

PBST washes in the well plate were removed as much as possible, and then 800. Mu.L of elution buffer (1 mg mL) was added to the cell culture flask ^-1 BSA,0.1M glycine, ph=2.2), incubated on ice for 15min, 200 μl Tris-HCl was added to the mixture to neutralize the eluate.

1.17, second to fourth round screening

The second, third and fourth rounds of screening were performed following the same procedure, with the phage solution after each round of amplification purification as the secondary phage peptide pool for the next round of screening. The phage input amount was kept consistent for each round of screening, and the titer of the eluted product was measured as 9.15X10% per round of phage output, respectively ⁴ ,3.04×10 ⁵ ,and 3.74×10 ⁵ pfu mL ^-1 And calculating the output/input ratio of each round of phage to be 4.58×10 respectively ^-7 ，1.52×10 ^-6 ，1.87×10 ^-6 。

The phage output results are shown in FIG. 1, and FIG. 1 is a graph showing the results of a three-round repeated screening experiment for gp140 protein-specific binding polypeptides using phage 7 peptide library. The experimental results show that the phage output increases progressively from round to round with the same phage input, indicating that phage with strong affinity for gp140 are effectively enriched.

1.18 phage-Positive monoclonal

The second, third and fourth rounds of eluted products were subjected to titer determination. Selecting plates with the number of plaques of 30-300, randomly picking 50 phage blueprint, adding the plates into 50 shaking tubes containing 5mL of LB culture medium, and carrying out shaking culture at 37 ℃ and 220rpm for 24 hours. mu.L of each monoclonal culture was added to an EP tube containing 300. Mu.L of glycerol (50%) solution, mixed well and stored in a-80℃refrigerator. And (5) sequencing the residual bacterial liquid.

Test 2: the DNA sequence of phage monoclonal was analyzed.

The amino acid sequence of the polypeptides was analyzed using DNAStar software, and binding sites for polypeptides and proteins were simulated using CABS-dock website.

The polypeptide has amino acid sequence comprising MHLHRTP sequence, WPHHHHHH sequence, WPFHHHHKH sequence, EWTLGNW sequence, FAWNMNP sequence, SHMALSP sequence, YFSPSFS sequence, FPTITPN sequence, WSPGYSG

Sequence, etc. 50 test sequences.

The results are shown in FIGS. 2 and 3. FIG. 2 shows the polypeptide sequences with positive monoclonal phage occurrence frequency greater than 2 and the number of repetitions. Wherein phage of the MHLHRTP polypeptide sequence appear 3 times, phage of the WPHHHHHH polypeptide sequence appear 3 times, and WPFHHHHHH polypeptide sequence appear 3 times.

FIG. 3 is a schematic representation of three polypeptide sequences in combination with gp140 protein binding sites.

Test 3: the affinity of the positive phage to gp140 protein was detected by phage enzyme-linked immunosorbent assay.

3.1 amplification and purification of Positive monoclonal phages

The E.coil.ER2738 strain frozen was removed from the-80℃refrigerator, a small amount of the ice residue was picked up by using a gun head and added to a shaking tube containing 6mL of LB medium (containing tetracycline), and shaking culture was carried out at 37℃at 220rpm overnight. 20mL of LB medium was placed in 5 250mL Erlenmeyer flasks, and after autoclaving, 500. Mu.L of overnight culture broth was added. The bacteria were grown in mid-log phase at 37℃with shaking at 220 rpm.

Frozen 3 positive monoclonal phage samples, 1 wild phage, 1 phage of random sequence group (polypeptide sequence is WSLGYTG) and 1 PBS blank are taken out from a refrigerator at-80 ℃, and 100 mu L of the frozen positive monoclonal phage samples are added into 5 conical flasks after the frozen positive monoclonal phage samples are melted. Standing at room temperature for 20min, placing into a shaking table at 37deg.C, and shaking at 220rpm for 4.5 hr. The culture broth was added to 5 50mL centrifuge tubes and centrifuged at 12000g for 20min. The supernatant was poured into 5 centrifuge tubes containing 4mL of PEG/NaCl solution, placed in a refrigerator at 4℃and allowed to settle overnight. The next day, the centrifuge tube 12000g was centrifuged for 20min, and the supernatant was discarded. 1mL of PBS solution was added to the centrifuge tube, the pellet was redissolved, and the resuspension was transferred to a 1.5mL EP tube, placed in a shaker at 37℃and shaken for 1 hour. The EP tube 12000g was then centrifuged for 10min, and the supernatant was transferred to an EP tube containing 200mL of PEG/NaCl solution. EP tubes were placed at 4℃and left to stand for 1 hour to settle phages. After that, the EP tube 12000g was centrifuged for 20min, and the supernatant was discarded, and 100. Mu.L of PBS was added to resuspend the pellet.

3.2 coating and sealing of Gp140 protein

Gp140 protein solution was added to 96-well plates and the 96-well plates were placed in wet cartridges. The wet box was placed on a rocker and coated overnight at 4 ℃. The liquid in the 96-well plate was washed using a pipette and the plate was spun down on sterile filter paper to thoroughly remove the liquid. 200. Mu.L of 0.5% BSA blocking solution was added. The well plate was placed on a rocker shaker and closed for 1 hour.

3.3 washing

The liquid in the 96-well plate was washed using a pipette and the plate was spun down on sterile filter paper to thoroughly remove the liquid. 1mL of PBST solution was added and the well plate was placed on a rocker shaker. After 5min, the liquid in the well plate was again pipetted and beaten, 1mL of PBST solution was added and the well plate was placed on a rocker shaker. This step was repeated 6 times to thoroughly wash phage in the well plate that bind or bind poorly to gp140 protein.

3.4 binding of Positive monoclonal phages to gp140 protein

5 phages were added at the same concentration to a 96-well plate and incubated at room temperature for 1 hour.

3.5 washing

The liquid in the 96-well plate was washed using a pipette and the plate was spun down on sterile filter paper to thoroughly remove the liquid. 1mL of PBST solution was added and the well plate was placed on a rocker shaker. After 5min, the liquid in the well plate was again pipetted and beaten, 1mL of PBST solution was added and the well plate was placed on a rocker shaker. This step was repeated 6 times to thoroughly wash phage in the well plate that did not bind or did not bind firmly to gp140 protein.

3.6 binding of primary antibody

Phage pVIII protein antibodies were added to 96-well plates and the plates were placed in a 37℃incubator for 1 hour.

3.7 washing

The liquid in the 96-well plate was washed using a pipette and the plate was spun down on sterile filter paper to thoroughly remove the liquid. 1mL of PBST solution was added and the well plate was placed on a rocker shaker. After 5min, the liquid in the well plate was again pipetted and beaten, 1mL of PBST solution was added and the well plate was placed on a rocker shaker. This step was repeated 6 times.

3.8 binding of secondary antibody

Secondary antibody of coupled horseradish peroxidase is added into a 96-well plate, and the plate is placed in a 37 ℃ incubator for standing for 1 hour.

3.9 washing

The liquid in the 96-well plate was washed using a pipette and the plate was spun down on sterile filter paper to thoroughly remove the liquid. 1mL of PBST solution was added and the well plate was placed on a rocker shaker. After 5min, the liquid in the well plate was again pipetted and beaten, 1mL of PBST solution was added and the well plate was placed on a rocker shaker. This step was repeated 3 times.

3.10, color development

Chromogenic substrate TMB was added to a 96-well plate and the plate was placed in an incubator at 37℃for 15min.

3.11 termination of

The reaction was stopped by adding 2M sulfuric acid solution to a 96-well plate.

3.12 measurement of results

Absorbance at 450nm was measured by means of an enzyme-labeled instrument. The results were saved and analyzed.

As a result, as shown in FIG. 4, the binding strength of the gp140 protein was higher for all 3 kinds of engineered phages than for wild-type phages (M13-WT), random sequence phages and Control group phages (Control), wherein phages displaying MHLHRTP sequences bound to gp140 protein were significantly stronger than for other phages.

The amino acid sequence related to the invention is as follows:

SEQ ID No.1；

name: amino acid sequence of polypeptide 1

The source is as follows: artificial sequence (Artificial Sequence)

MHLHRTP

SEQ ID No.2；

Name: amino acid sequence of polypeptide 2

The source is as follows: artificial sequence (Artificial Sequence)

WPHHRHH

SEQ ID No.3；

Name: amino acid sequence of polypeptide 3

The source is as follows: artificial sequence (Artificial Sequence)

WPFHHKH。

Claims

1. A polypeptide targeting gp140 protein, which is characterized in that the amino acid sequence of the polypeptide is one of the amino acid sequences shown in No. 1-No. 3.

2. A bioactive substance characterized by: comprising the polypeptide of claim 1, further comprising a covalently linked compound or nanoparticle, an engineered phage, or other microorganism, a mixture of polymers.

3. A polynucleotide sequence capable of encoding any one of the amino acid sequences of claim 1 or capable of encoding the biologically active substance of claim 2.

4. A polypeptide for use in the treatment of a disease comprising the polypeptide of claim 1.

5. The use of the polypeptide of claim 1, the biologically active fragment of claim 2, the polynucleotide sequence of claim 3, the polypeptide drug of claim 4, characterized in that: the application of the composition in preparing biomedical materials and medicines for HIV antiviral treatment.

6. The use according to claim 5, characterized in that: use in the preparation of biomedical materials and medicaments for the realization of HIV antiviral therapy by targeted binding to gp140 protein.