CN116478242A - Phage polypeptide for targeting binding to novel coronavirus receptor binding region and application thereof - Google Patents

Abstract

The invention provides a phage polypeptide targeting the binding region of the novel coronavirus receptor and its use, belonging to the field of biotechnology. The present invention uses phage display technology to screen the phage-displayed dodecapeptide library by using the novel coronavirus receptor binding region protein as a target molecule, and obtains a phage polypeptide targeting the novel coronavirus receptor binding region protein, whose amino acid sequence is: SerValTyrAsnAlaLeuTyrLeuAsnAlaAlaGlu. The above polypeptides have good binding properties and specificity to the novel coronavirus receptor binding region protein. The polypeptide of the present invention can be applied in the fields of analysis and detection of novel coronavirus, development of polypeptide drugs, capture and release of novel coronavirus, etc., and provides components for immune analysis and therapeutic drugs of novel coronavirus.

Description

A phage polypeptide targeting the binding region of the novel coronavirus receptor and its use

technical field

The invention belongs to the field of biotechnology, and in particular relates to a phage polypeptide that binds to a protein in a receptor binding region of the novel coronavirus.

technical background

The disease caused by the new coronavirus (SARS-CoV-2) can be transmitted through crowds or environmental media. It is highly pathogenic and contagious, and has become one of the most severe challenges in human history, posing a huge threat to people's lives and health, public health systems and economic development.

The new type of coronavirus (SARS-COV-2), the particles are round or oval, with a diameter of 60-140nm, belonging to the βcoronavirus genus, including non-structural proteins (orf 1\ab encoding) and structural proteins (N: nucleocapsid protein, S: spike glycoprotein, E: envelope protein, M: membrane protein). In the process of SARS-COV-2 entering the host, the Receptor-Binding Domain (RBD, 319aa-541aa) protein on the spike glycoprotein plays a crucial role and is a bridge for the virus to enter the host. Therefore, the RBD protein is usually regarded as an excellent detection and treatment target. The novel coronavirus RBD monoclonal antibody is often used as a detection element or therapeutic drug for the novel coronavirus. For example, the patent "An anti-new coronavirus monoclonal antibody and its application" (application number: CN202010151348.6) discloses a novel coronavirus RBD protein monoclonal antibody that can be applied to the diagnosis, prevention and treatment of the novel coronavirus. However, the monoclonal antibody has the disadvantages of high preparation cost, long cycle, professional operators, antigen consumption, and large batch-to-batch differences. Therefore, it is necessary to find its replacement components.

Phage display technology was invented by Smith in 1985 and won the Nobel Prize in Chemistry in 2018. The principle of this technology is to insert a fragment of a foreign gene (encoding polypeptide or protein) into the coat protein gene of a filamentous phage. The foreign gene will be displayed on the surface of the phage along with the expression of the coat protein, maintaining independent biological activity and spatial conformation to facilitate the recognition and binding of the target protein. The phage random display peptide library is put into the solid phase carrier coated with the target substance, and after a period of incubation, the unbound or weak affinity phage is washed away, and the bound phage is obtained by competitive elution or acid elution. According to this step, 3-5 rounds of screening are performed to obtain phage that can bind to the target characteristic. Since the first report, this technology has been widely used in the screening of monoclonal antibodies, macromolecular proteins, small molecules, enzymes and other substances. Phage-displayed peptides have the advantages of low preparation cost, short time, and small batch-to-batch variation, and are excellent substitutes for monoclonal antibodies, toxin antigens, and other substances.

Contents of the invention

In view of the shortcomings of traditional monoclonal antibodies, the purpose of the present invention is to provide phage polypeptides that specifically bind to the new coronavirus receptor binding region protein, so as to be used as a substitute for traditional RBD monoclonal antibodies for the detection and treatment of new coronavirus.

The object of the present invention is implemented through the following technical solutions:

(1) Coat the purchased novel coronavirus RBD protein on enzyme-labeled wells, seal with 3-5% skimmed milk powder for two hours, add a phage display random dodecapeptide library, apply PBST to absorb phages that do not bind or have weak binding force, acid-elute the target phages, and then amplify the eluted phages to a certain titer as the peptide library for the next round of screening. Three rounds of screening were carried out according to the steps of "adsorption-washing-elution-amplification". During the screening process, the concentration of PBST, phage incubation time, acid elution time and other conditions were changed to make the screening conditions gradually harsher in order to screen for phages with stronger affinity and specificity.

(2) After three rounds of screening, 46 monoclonal phages were selected for preliminary identification by phage-ELISA. For the phages that can bind to the receptor binding region protein of the novel coronavirus, bovine serum albumin, chicken ovalbumin, maltose protein, human soluble growth-stimulating expression protein, anti-fumonisin nanobody, and human adiponectin protein were used as negative controls, and PBS was used as a blank control to obtain two phages that specifically bind to the receptor binding region protein. The two phages were amplified, plasmid extracted, Sequencing found that the inserted polypeptide chain of one of the phages was missing, and the inserted polypeptide sequence of the other phage was S-V-Y-N-A-L-Y-L-N-A-A-S-E. The polypeptide consisted of 12 amino acids and was inserted on the coat protein gene of the phage.

The phage display polypeptide of the present invention can specifically bind to the new coronavirus receptor binding region protein, and as a replacement element of the traditional RBD monoclonal antibody, it can be applied to the detection and treatment of the new coronavirus.

The present invention has the following benefits:

(1) Compared with traditional monoclonal antibodies, the phage-displayed polypeptide of the present invention has the advantages of simple operation, low cost, easy preparation, easy modification, and high biological safety.

(2) The polypeptide sequence of the present invention is reported for the first time at home and abroad, and has high innovation.

(3) The phage display polypeptide provided by the present invention can be applied to products such as probes or kits used for immunoassay or treatment of novel coronavirus.

Description of drawings

Figure 1 is the result of selecting 46 phage clones and verifying positive clones by Phage-ELISA; the abscissa is the number of the phage clone, and the ordinate is the absorbance at 450nm;

Fig. 2 is the result of verifying the specificity of 3-22 phage by Phage-ELISA; the abscissa is the type of protein coated on the microtiter plate; the ordinate is the absorbance value at 450nm.

Detailed ways

Materials used, reagents used and formula are as follows in the embodiment of the present invention:

Main experimental materials:

The novel coronavirus receptor binding region protein (Ningbo Maiyue), phage random display dodecapeptide library, and E.coilER2738 are preserved by the Food Quality and Safety Laboratory of the Sino-German Joint Research Institute of Nanchang University.

Main reagents:

HRP (horseradish peroxidase) enzyme-anti-phage monoclonal antibody (Beijing Yiqiao Shenzhou Technology Co., Ltd.), Tween-20 (Beijing Suolaibao Technology Co., Ltd.), chicken ovalbumin (Beijing Suolaibao Technology Co., Ltd.), bovine ovalbumin (US Sigma Company), skim milk powder (Shanghai Sangong Biotechnology Co., Ltd.), tetramethylbenzidine (Shanghai Sangon Biotechnology Co., Ltd.), isopropyl-β-D-thiogalactoside (Shanghai Sangon Biotechnology Co., Ltd.), 5-bromo- 4 Chloro-3-indole-β-D-galactoside (Tiangen Biochemical Technology Co., Ltd.), Tetracycline (Beijing Suolaibao Technology Co., Ltd.), PEG8000 (Beijing Suolaibao Technology Co., Ltd.), LB Broth (Qingdao Haibo Biotechnology Co., Ltd.)

Main reagent formula:

1. LB liquid medium: Weigh 25g LB broth and dissolve it in 1000mL ultrapure water, autoclave at 121℃ for 15min;

2. LB/IPTG/X-gal plate: LB solid medium, autoclaved, add 1% IPTG/X-gal and mix well, pour 25mL into each sterilized plate, cool and store at 4°C, sealed and protected from light;

3. Top layer agar: LB liquid medium, 1g/LMgCl ₂ 6H ₂ O, 7.5g/L agar powder, 4mL/tube, autoclave for 15min;

4. 20% PEG-NaCl: 50g PEG-8000, 36g NaCl, heated to dissolve in ultrapure water, dilute to 250mL, autoclave for 15min;

5. Eluent: 0.2M Gly-HCl, adjust pH=2.2, autoclave for 15 minutes;

6. Neutralization buffer: 1M Tris-HCl, adjust pH=9.1, autoclave for 15 minutes;

7. TBS buffer: 50mM Tris-Hcl, 150mM NaCl, autoclave for 15min;

8. 0.1%, 0.25%, 0.5% TBST buffer: 1000 μL, 2500 μL, 5000 μL of Tween-20 was added to 1 LTBS buffer, and autoclaved for 15 minutes;

Screening and identification of novel coronavirus receptor binding domain (RBD) protein phage polypeptides:

1. Screening of bacteriophage polypeptides for the receptor binding region protein of the novel coronavirus

According to the steps of "coating-adsorption-washing-elution-amplification-identification", three rounds of screening were carried out

(1) Wet the 96-well ELISA plate with sterile ultrapure water, place it in an ultra-clean workbench, and irradiate it with ultraviolet light for half an hour to sterilize;

(2) Take 100 μL of RBD protein with a concentration of 50 μg/mL to the microtiter plate sterilized by ultraviolet light, and wrap it in a refrigerator at 4°C overnight;

(3) In a sterile operating bench, wash with sterile 0.1% TBST washing solution three times, pat dry each time on sterile paper, then add 250-350 μL of 1-3% BSA-TBS blocking solution sterilized by filtration, and seal in a 37°C incubator for 1-2h;

(4) Wash three times with sterile 0.1% TBST washing solution, pat dry each time on sterile paper, take the dodecapeptide library (2×10 ¹¹ pfu/well), add 100 μL sterile 1×TBS, mix well, add to the above microtiter plate, and combine at 37°C for 1 hour;

(5) Wash three times with 0.1% TBST, add 100 μL of Gly-HCl elution buffer, shake at a constant speed at 37°C for 8-15 minutes, suck out the eluted product, and quickly add a pre-adjusted appropriate volume of Tris-HCl neutralization buffer until neutral;

(6) Take 10-15 μL for the determination of phage titer, and use the rest for phage amplification.

(7) Streak inoculation of E.coli ER2738 on LB/Tet solid medium plate, culture at 37°C for 12-16h;

(8) Pick a monoclonal colony from the plate and inoculate it into a test tube of LB/Tet liquid medium, and culture it with shaking at 37°C and 220rpm for 12-16h;

(9) Pipette 100-500 μL of the above-mentioned culture into 40-60 mL LB/Tet liquid medium, add the eluted phage, shake and culture at 37°C and 220 rpm for 4-6 hours;

(10) Transfer the amplified product to a sterilized centrifuge tube, centrifuge at 4°C and 8000rpm for 10-15min, and collect the supernatant into a fresh sterilized centrifuge tube;

(11) Add 8-10 mL (1/6 of the volume of the supernatant) sterile 10-20% PEG-NaCl solution to the supernatant, shake well, and let stand at 4°C for 12-16 hours;

(12) Centrifuge at 4°C and 8000rpm for 10-15min, discard the supernatant, resuspend the pellet with 1mL sterile PBS buffer, transfer to a sterile centrifuge tube, add 200μL 20% PEG-NaCl solution, mix well, and ice-bath for 2h;

(13) Centrifuge at 4°C and 12000rpm for 10-15min, discard the supernatant, then centrifuge briefly, absorb the remaining supernatant, resuspend the pellet with 200μL sterile PBS, and centrifuge at 4°C and 5000rpm for 1-2min;

(14) Transfer the supernatant to a fresh centrifuge tube. Take 10 μL to measure the phage titer and store at -20°C;

(15) Steps (1)-(14) are the first round of amplification process, the second round and the third round of panning steps are roughly the same, the amount of phage input in each round is 2×10 ¹¹ pfu, the coating concentration of RBD is reduced to 30 μg/mL and 10 μg/mL, respectively, 1-3% OVA-TBS and 1-3% BSA-TBS blocking solution are alternately blocked, and the binding time of input phage and RBD protein is 45min and 30min respectively , the concentrations of the washing solutions were 0.25% TBST and 0.5% TBST, respectively. The panning scheme is shown in Table 1.

Table 1 Screening process of novel coronavirus receptor binding region protein phage polypeptide

Determination of phage titer

(1) Streak inoculation of E.coli ER2738 on LB/Tet solid medium plate, culture at 37°C for 12-16h;

(2) Pick a monoclonal colony from the plate and inoculate it into LB/Tet liquid medium, culture at 37°C with shaking at 220rpm until the logarithmic growth phase (OD _600nm ≈0.5-0.6);

(3) Take 200 μL of the bacterial solution in (2) into a sterile 1.5mL centrifuge tube;

(4) Dilute the eluted phage to 10 ^-2 , 10 ^-3 , 10 ^-4 times, put the top layer of agar in a microwave oven to heat and melt;

(5) Add 10 μL of phage solutions with different dilutions to the centrifuge tube in (4), mix well, and incubate at 37°C for 10-15 minutes;

(6) Inject the bacteriophage-infected bacterial solution into the top agar, shake slightly to mix, quickly pour it onto the LB/IPTG/X-gal plate, and incubate at 37°C for 12-16h;

(7) Pick a plate with a length of about 30-300 blue spots and count them, and calculate the phage titer. The calculation formula is: colony forming unit (cfu) = number of colonies in the plate × dilution factor × 100.

2. Screening and identification of positive clones

After three rounds of screening, a plate with about 200 blue spots was picked, and 46 clones were selected for amplification and phage-ELISA identification. The specific operation steps are as follows:

(1) Pick a single colony clone and inoculate in LB/Tet liquid medium, culture at 37°C and 220rpm for 12-16h;

(2) Take 500 μL of overnight E.coil ER2738 culture into 50 mL of liquid medium, and mix well;

(3) Dispense 1 mL of the culture medium in (2) into centrifuge tubes;

(4) Pick a single blue phage plaque into each centrifuge tube, 37°C, 220rpm shaking culture for 4.5-6h;

(5) After the culture is over, centrifuge at 8000rpm for 2min, draw the supernatant to a fresh centrifuge tube, and mark it with a number. Stand at 4°C for later use.

(6) Take 100 μL of RBD protein at a concentration of 1 μg/mL from each well and coat it on a microtiter plate, overnight at 4°C;

(7) Wash the plate three times with 0.05% PBST, add 300 μL of 5% skimmed milk to block, and incubate at 37°C for 1-2 hours;

(8) Wash the plate three times with 0.05% PBST, add 100 μL of plaque supernatant culture solution, and incubate at 37°C for 45-60min;

(9) Wash the plate three times with 0.05% PBST, add 100 μL anti-M13 secondary antibody to each well, and incubate at 37°C for 45-60 minutes;

(10) Wash the plate three times with 0.05% PBST, add 100 μL of TMB chromogenic solution, and incubate at 37°C for 8-15 minutes;

(11) Add 50 μL of 2M H ₂ SO ₄ to each well, and measure the OD _{450 nm} absorbance value. Accompanying drawing 1 shows the result of selecting 46 phage clones and verifying positive clones by Phage-ELISA; the abscissa is the numbering of the phage clone, and the ordinate is the absorbance at 450nm;

(12) Among the 46 clones selected, 16 clones could bind to RBD protein, and positive clones were selected for further specificity verification.

Specificity of positive clones identified by Phage-ELISA:

(1) 100 μL of RBD protein with a concentration of 1 μg/mL, 1-3 μg/mL bovine serum albumin (BSA) protein, chicken ovalbumin (OVA) protein, maltose (MBP) protein, human soluble growth-stimulating expression (SST2) protein, anti-fumonisin B ₁ (FB ₁ ) nanobody, and human adiponectin (gAd) protein were coated onto the microtiter plate, and three groups of parallel controls were set up, overnight at 4°C;

(2) The steps are the same as (7)-(11) in the identification of positive clones;

(3) Among the 13 positive phage plaques identified, 3-22 phage plaques showed better affinity and specificity (Fig. 2).

Specific application of the present invention: the present invention specifically relates to a phage polypeptide that binds to the protein of the novel coronavirus receptor binding region, which can be used as a detection element through in vitro expression technology to develop a novel coronavirus analysis system and detection kit based on immunohomogeneous, immunological test strips, microfluidics and other detection means.

The above-mentioned embodiments are implementation methods of the present invention. For those skilled in the art, without departing from the concept of the present invention, some applications and improvements can be made, but all belong to the protection scope of the present invention.

Claims (7)

1. A phage polypeptide targeting the binding region of the novel coronavirus receptor, characterized in that the polypeptide has the amino acid sequence of SEQ ID NO:1. 2. The phage polypeptide targeting the binding region of the novel coronavirus receptor according to claim 1, characterized in that it consists of 12 amino acids and has good binding performance and specificity to the RBD protein of the receptor binding region of the novel coronavirus. 3. The phage polypeptide targeting the binding region of the novel coronavirus receptor according to claim 1 or 2, characterized in that it is connected to the M13 phage coat protein by GGGSS. 4. The method for preparing the phage polypeptide targeting the binding region of the novel coronavirus receptor according to any one of claims 1-3, characterized in that it mainly comprises the following steps: (1) The novel coronavirus RBD protein was used as a screening target and put into a phage display 12-peptide library for three rounds of solid-phase screening. (2) The monoclonal phage plaque amplification after three rounds of screening was randomly selected. (3) Phage-ELISA identifies the binding ability and specificity of the RBD protein, and the plaques that bind to the novel coronavirus RBD protein will be sequenced. 5. The method for preparing a phage polypeptide targeted for binding to a novel coronavirus receptor binding region according to claim 4, wherein the first round of screening amplification step is: (1) Wet the 96-well ELISA plate with sterile ultrapure water, place it in an ultra-clean workbench, and irradiate it with ultraviolet light for half an hour to sterilize; (2) Take 100 μL of RBD protein with a concentration of 50 μg/mL to the microtiter plate sterilized by ultraviolet light, and wrap it in a refrigerator at 4°C overnight; (3) In a sterile operating bench, wash with sterile 0.1% TBST washing solution three times, pat dry each time on sterile paper, then add 250-350 μL of 1-3% BSA-TBS blocking solution sterilized by filtration, and seal in a 37°C incubator for 1-2h; (4) Wash three times with sterile 0.1% TBST washing solution, pat dry each time on sterile paper, take the dodecapeptide library (2×10 ¹¹ pfu/well), add 100 μL sterile 1×TBS, mix well, add to the above microtiter plate, and combine at 37°C for 1 hour; (5) Wash three times with 0.1% TBST, add 100 μL of Gly-HCl elution buffer, shake at a constant speed at 37°C for 8-15 minutes, suck out the eluted product, and quickly add a pre-adjusted appropriate volume of Tris-HCl neutralization buffer until neutral; (6) Take 10-15 μL for the determination of phage titer, and all the rest are used for the amplification of phage; (7) Streak inoculation of E.coli ER2738 on LB/Tet solid medium plate, culture at 37°C for 12-16h; (8) Pick a monoclonal colony from the plate and inoculate it into a test tube of LB/Tet liquid medium, and culture it with shaking at 37°C and 220rpm for 12-16h; (9) Pipette 100-500 μL of the above-mentioned culture into 40-60 mL LB/Tet liquid medium, add the eluted phage, shake and culture at 37°C and 220 rpm for 4-6 hours; (10) Transfer the amplified product to a sterilized centrifuge tube, centrifuge at 4°C and 8000rpm for 10-15min, and collect the supernatant into a fresh sterilized centrifuge tube; (11) Add 1/6 supernatant volume of sterile 10-20% PEG-NaCl solution to the supernatant, shake well, and let stand at 4°C for 12-16h; (12) Centrifuge at 4°C and 8000rpm for 10-15min, discard the supernatant, resuspend the pellet with 1mL sterile PBS buffer, transfer to a sterile centrifuge tube, add 200μL 20% PEG-NaCl solution, mix well, and ice-bath for 2h; (13) Centrifuge at 4°C and 12000rpm for 10-15min, discard the supernatant, then centrifuge briefly, absorb the remaining supernatant, resuspend the pellet with 200μL sterile PBS, and centrifuge at 4°C and 5000rpm for 1-2min; Transfer the supernatant to a fresh centrifuge tube, take 10 μL to measure the phage titer, and store at -20°C. 6. The method for preparing a phage polypeptide targeting the receptor binding region of the novel coronavirus according to claim 4, wherein, in the second round and the third round of screening, the coating concentration of RBD is reduced to 30 μg/mL and 10 μg/mL respectively, and 1-3% OVA-TBS and 1-3% BSA-TBS blocking solution are used to alternately block, and the binding time of phage and RBD protein is respectively 45min and 30min, and the concentration of washing solution is 0.25% TBST and 0 respectively. .5% TBST; the amount of phage input in each round was 2×10 ¹¹ pfu. 7. The use of the phage polypeptide targeting the binding region of the new coronavirus receptor according to any one of claims 1-3, characterized in that it is used to prepare products that can be applied to analyze or neutralize the new coronavirus.