CN107793471B

CN107793471B - Folate receptor alpha specific binding peptide 4 and application thereof

Info

Publication number: CN107793471B
Application number: CN201710649314.8A
Authority: CN
Inventors: 王旻; 邱郑; 徐祎凤; 邢黎军; 王红
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2017-07-26
Filing date: 2017-07-26
Publication date: 2020-08-11
Anticipated expiration: 2037-07-26
Also published as: CN107793471A

Abstract

The invention relates to a short peptide specifically combined with folate receptor alpha and application thereof, belonging to the technical field of biology. The polypeptide sequence of the invention is YTNPYYSSHTRN. The polypeptide is obtained by screening from a phage display library, can be specifically combined with tumor cells expressing folate receptor alpha, provides a new targeting molecule for targeted therapy of tumors and tumor images, and has potential value of new drug development.

Description

Folate receptor alpha specific binding peptide 4 and application thereof

The technical field is as follows:

the invention belongs to the technical field of biology, and relates to a short peptide specifically bound with folate receptor alpha and application thereof in tumor targeting.

Background art:

the phage display technology is to perform fusion expression on exogenous polypeptide or protein and a certain capsid protein of a phage, so that the exogenous protein can be displayed on the surface of a virus particle, and simultaneously, DNA encoding the exogenous protein is positioned in the virus particle. The random dodecapeptide phage display library is a combined library formed by fusing random dodecapeptide to M13 phage minor capsid protein (pIII). The displayed dodecapeptide is expressed at the N-terminus of pIII. This creates a direct bridge between a large number of random polypeptides and their DNA coding sequences, and then in vitro screening is performed with various target molecules (antibodies, enzymes, cell surface receptors, etc.) to obtain target molecule binding peptides. The in vitro selection procedure is simply the co-incubation of a phage library with a solid phase target molecule, washing to remove unbound phage, and then eluting to obtain phage capable of specifically binding to the target molecule. The eluted phage is then amplified for the next binding/amplification cycle to enrich for specifically binding phage. After 3-4 rounds of "panning", each specifically bound polypeptide sequence can be obtained by DNA sequencing.

Folate Receptors (FRs) are important transporters that bind and transport folate and its derivatives into cells, and exist in vivo primarily as three subtypes: FR α, β and γ. Folate receptor alpha (FR α) is a glycoprotein anchored to the surface of cell membranes by Glycosylated Phosphatidylinositol (GPI). The FR alpha in tissues such as ovarian cancer, lung cancer, liver cancer, breast cancer and the like is high in expression and limited in expression in normal tissues, so that the FR alpha is considered as an ovarian cancer marker or Tumor Associated Antigen (TAA) with great potential; the FR alpha has high specificity to ovarian cancer and can be used as a target for treating related tumors; some studies have also shown that FR α is of great value for the early diagnosis of ovarian cancer. Based on the above, if a short peptide sequence with higher affinity with the FR alpha can be obtained, a new idea is provided for targeted therapy and diagnosis of the tumor.

Disclosure of Invention

Object of the Invention

The invention aims to obtain a polypeptide capable of being specifically combined with a folate receptor alpha by utilizing a phage display technology. The polypeptide can be used for targeting tumor cells positive in folate receptor alpha expression.

Technical scheme

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

1) affinity screening of phage display peptide libraries: the folic acid receptor alpha recombinant protein is used as a target, and four rounds of biological screening are carried out by using a phage display dodecapeptide library. Detecting recovery rate and polyclonal ELISA by each screening to judge whether the screening is effective; detecting the binding capacity of each clone and the folate receptor alpha recombinant protein by monoclonal ELISA;

2) preparing phage positive monoclonal DNA and sequencing and identifying: selecting phage monoclone with high positive value according to the ELISA result, amplifying the phage monoclone, quickly purifying a sequencing module to generate a sufficiently pure template for sequencing, selecting-96 gIII sequencing primers to send to a company for sequencing, and analyzing a corresponding display peptide sequence of the positive phage;

3) cell ELISA detects binding of phage clones to native cell surface folate receptor alpha: selecting an FR alpha expression positive cell strain SKOV3, and detecting the combination of phage clone and cell strain by ELISA by taking an FR alpha expression negative cell strain HepG2 as a control;

4) the binding of the phage clones to the FR α expression positive cell line SKOV3 was further confirmed by flow cytometry.

Advantageous effects

The FR alpha binding peptide provided by the invention can be specifically bound with FR alpha recombinant protein, and meanwhile, through cell ELISA and flow cytometry experimental analysis, the screened phage display short peptide can be specifically bound with natural FR alpha on the surface of a cell, so that the FR alpha binding peptide has potential medical and pharmaceutical values.

Description of the drawings:

FIG. 1 polyclonal ELISA;

FIG. 2 is a graph showing the study of the binding ability of positive monoclonals to folate receptor alpha recombinant proteins. FIG. 2A. monoclonal phage ELISA screening positive clones (the circled clones are folate receptor alpha specific binding peptide 4 display clones); FIG. 2B further validation of binding of positive monoclonals to folate receptor alpha recombinant protein;

FIG. 3 shows that the positive monoclonal antibody screened by ELISA is combined with SKOV3, which is a FR alpha expression positive cell, and M13 and HepG2, which is a FR alpha expression negative cell, are used as controls;

FIG. 4. flow cytometry analysis of the specific binding of positive monoclonal antibodies to SKOV3, for the control group, only SKOV3 cells with anti-M13 antibody and FITC fluorescent antibody were added, as indicated by the dashed line in the figure.

The specific implementation mode is as follows:

while the following provides more particular embodiments for purposes of illustrating the invention more clearly, it will be understood by those skilled in the art that the present invention is not limited to the examples set forth below.

Example 1 affinity screening of phage display peptide libraries

Random dodecapeptide phage display libraries were purchased from NEB, 100. mu.l, with titers of 1.5 × 10¹³pfu/ml. Stored in 50% glycerol in TBS buffer (50mM Tris-HCl, 150mM NaCl [ pH7.5 ]]) Medium, reservoir capacity 2.7 × 10⁹And (4) a transformant. Escherichia coil ER2738 is the host bacterium for the peptide library.

(1) Preparation work

The FR α recombinant protein was diluted with a coating buffer (carbonate buffer [ pH9.6]) to a final concentration of 50 μ g/ml, and the diluted solution was coated on an ELISA plate at 100 μ l per well, and the rotation was repeated until the surface was completely wetted. Incubate overnight at 4 ℃ in a wet box.

(2) Affinity screening

The next day, the coating solution was poured off the microplate and applied to TBST buffer (TBS + 0.1% by volume [ v/v ]]Tween-20) plates were washed, buffer was decanted, and residual liquid was removed by patting on clean absorbent paper. Then 200. mu.l of blocking solution (0.1M NaHCO) was added to each well₃，5mg/ml BSA，0.02％NaN₃[PH8.6]) Putting the mixture into a wet box, incubating the mixture at 4 ℃ overnight or incubating the mixture at 37 ℃ for at least 1 hour, discarding the blocking solution, filling TBST buffer solution into each hole, washing the plate for 6 times, washing the plate for 5 minutes each time, putting the plate on a decoloration shaking bed for carrying out washing, then pouring the buffer solution, beating and throwing the buffer solution on clean absorbent paper to remove residual liquid, quickly carrying out the experimental step to avoid drying the hole plate, diluting the original library by using the TBST buffer solution, taking 100 mu l of the diluted library solution, adding the library solution into the micropore of the ELISA plate which is coated with FR α recombinant protein in advance, wherein the quantity of the added phage is about 5 × 10¹². Incubate overnight at 4 ℃ or 2 hours at 37 ℃ in a wet box, discard the well contents, flap the plate upside down to remove residual solution, and wash the plate 10 times with TBST buffer, as before (wash away unbound phage). After the final wash, the well is patted clean, and 100. mu.l of a nonspecific buffer such as 0.2M Glycine-Glycine hydrochloride-HCl buffer [ pH2.2 ] is added to the wells of the microplate]To elute the bound phage, gently shaking on a decolorizing shaker for 10 minutes at room temperature to allow for sufficient elution, followed by 15. mu.l of 1M Tris-HCl [ pH9.1 ]]The eluate was neutralized and transferred to a clean EP tube. Taking 5 μ l of eluent by conventional M13 method for determining eluate titer, adding the rest eluent into 20ml of ER2738 strain in early log phase for first round phage eluate amplification, shake culturing at 37 deg.C and 220rpm for 4.5 hr, transferring the amplified product into a clean centrifuge tube, centrifuging at 4 deg.C and 10,000rpm for 10 min, taking about 80% of phage supernatant, transferring into another clean centrifuge tube, adding 1/6 volume of PEG/NaCl (20% [ w/v ])]PEG-800，2.5M NaCl)Phage precipitation was performed by standing at 4 ℃ for at least 1 hour or overnight. Centrifugation was carried out at 10,000rpm for 10 minutes at 4 ℃ and the residual supernatant was discarded by a micropipette, and the supernatant was thoroughly aspirated by brief centrifugation. The pellet was resuspended in 200. mu.l TBS buffer, centrifuged again and the supernatant was transferred to a clean centrifuge tube, the amplified eluate. Mu.l of the eluate was taken for determination of phage titer, and the remainder was used for the second round of affinity screening. The steps are repeated for four rounds of screening. Round-by-round screening increased the number of washes of TBST in the wash step.

(3) Determination of phage titer

Inoculating ER2738 strain in 10ml LB culture medium, shake culturing at 37 deg.C to middle logarithmic phase (OD)₆₀₀Around 0.5). During the process, the upper layer agar is heated and dissolved in a microwave oven, and is subpackaged into 5ml sterilized EP tubes, wherein each tube is 3ml, the number of the tubes is determined according to the dilution gradient of the phage, and only one tube is needed for each dilution gradient. The subpackaged EP pipes are placed in a water bath kettle at 45 ℃ for standby; meanwhile, LB/IPTG/Xgal culture plates were prepared and placed in a 37 ℃ incubator for use. The collected phage supernatant was diluted with LB medium in a 10-fold gradient (general dilution range of amplified phage: 10)⁸～10¹¹) After dilution, 10 mul of diluted matters in various gradients are immediately added into a plurality of 1.5ml sterilized EP tubes, and each tube is 200 mul, the diluted matters in various gradients are immediately added into the EP tubes containing bacterial liquid, after shaking and mixing, incubation is carried out for 5 minutes at 37 ℃, then upper agar placed at 45 ℃ is taken out, the contents in the tubes are immediately and completely transferred into upper agar, the tubes are quickly inverted and mixed, poured onto pre-warmed LB/IPTG/Xgal culture plates, the plates are gently shaken to evenly distribute the upper agar, after cooling and solidification for a period of time, inverted culture is carried out at 37 ℃ for overnight, plates with the total number of the plaques being about 100 are selected, the number of the plaques growing on the plates is counted, the titer (pfu) of the phage is calculated, the quantity of the phage screened in each round (Input titer Input), the quantity of the eluted phage (Output titer Output), the Output ratio of each round is calculated, the enrichment degree reflecting the specificity (enrichment degree) of the Recovery, and the enrichment degree of the recombined proteins screened by four rounds of specificity α binding affinity binding with the FR, and the recombined proteins are subjected to be subjected to high affinity screening, and the FR is calculatedThe force phages were effectively enriched (see table 1).

TABLE 1 Titers, Titers exported and recoveries from each round

Example 2 phage polyclonal ELISA identification

FR α recombinant protein was treated with carbonate buffer [ pH9.6]Diluted to a final concentration of 10. mu.g/ml, coated with 100. mu.l per well, overnight at 4 ℃. Discarding the target molecule solution every other day, beating and throwing on clean absorbent paper to remove residual liquid, drying, washing once with TBS buffer solution, adding 200 μ l of blocking solution into each hole, and standing at 37 ℃ for 1-2 hours. The blocking solution was spun off, washed 3 times with TBS buffer 5 minutes each time, patted vigorously on clean absorbent paper, and the wash was spun off. After washing, 100. mu.l of diluted eluate (i.e., phage supernatant) from each round of screening amplification was added to each well, incubated at 37 ℃ for 2 hours, the well liquid was discarded, and washed with TBST buffer and TBS buffer for 5 minutes each time, after removing the washing liquid by beating, the HRP-labeled mouse anti-M13 antibody was added as a secondary antibody (diluted to working concentration with blocking solution), incubated at 37 ℃ for 2 hours, the well liquid was discarded, and the washing operation was the same as the above. Adding 100 mul TMB substrate solution into each hole for developing, incubating for 10 minutes at room temperature in a dark place, and changing the liquid in the holes from colorless to blue; add 50. mu.l of 1M H per well₂SO₄Stopping the color reaction in a stopping solution, and detecting OD on a microplate reader₄₅₀The value is obtained.

The results showed that the affinity of the eluate for binding to the FR α recombinant protein increased gradually as the number of screening rounds increased, but decreased somewhat at the fourth round (see fig. 1).

Example 3 ELISA and sequencing identification of phage monoclonals

(1) ELISA (enzyme-Linked immuno sorbent assay) for detecting binding capacity of monoclonal phage display short peptide to target molecule

Obtaining the phage to be detected: the strain ER2738 was inoculated in advance, cultured at 37 ℃ to the pre-log stage, and the pre-log culture was transferred to a 2ml deep well plate at 600. mu.l per well. Plates for titer detection (plates with a total of about 100 plaques and less) were selected from four rounds of screening, and a plurality of blue plaques were randomly picked with a tip into a deep well plate and incubated with a shaker at 37 ℃ and 220rpm for 4.5 hours. Centrifugation was carried out at 10,000rpm for 10 minutes at 4 ℃ to discard the precipitate, 100. mu.l of the supernatant per well was used for ELISA detection, and the remaining supernatants were collected and stored at 4 ℃.

ELISA detection by dissolving the target FR α recombinant protein at a final concentration of 10. mu.g/ml in carbonate buffer [ pH9.6]In each well, 100. mu.l was coated overnight at 4 ℃ in a sealed wet box. Throwing out the redundant target molecule solution, beating and throwing away the redundant liquid on a clean paper towel, adding 200 mu l of sealing liquid into each hole, and sealing for 1-2 hours at 37 ℃. Throwing out the blocking liquid, washing for 6 times by TBST, beating and drying the enzyme label plate, sequentially adding 100 mu l of supernatant obtained from the deep-hole plate, reacting for 1-2 hours at room temperature, washing for 6 times by TBST (the same operation is carried out), diluting HRP-labeled anti-M13 antibody by the blocking liquid according to the proportion of 1: 10000, adding 100 mu l of blocking liquid into each hole, reacting for 1-2 hours at room temperature, fully washing for 6 times by TBST (the same step is carried out), adding 100 mu l of TMB substrate liquid (used in preparation) into each hole for color development, adding 50 mu l of 1M H into each hole after 10 minutes of light shielding effect, and then adding 50 mu l of TMB substrate liquid (used in preparation) into each hole₂SO₄Stopping the color reaction in a stopping solution, and detecting OD on a microplate reader₄₅₀The value is obtained.

The results are shown in FIG. 2. Fig. 2A shows the preliminary detection results (the clone circled in fig. 2A is a folate receptor alpha specific binding peptide 4 display clone), and the clone is picked alone and verified to be capable of binding to the FR alpha recombinant protein through further ELISA experiments (fig. 2B), wherein PBS is used as a blank control, and the experimental group has a significant difference (P < 0.05) compared with the PBS control group.

(2) Preparation and sequencing identification of phage positive monoclonal DNA

According to the above ELISA results, a single positive phage clone was selected for amplification and then subjected to rapid purification of the sequencing module to generate a sufficiently pure template for sequencing: the overnight ER2738 culture was inoculated into LB medium at a ratio of 1: 100 and shaken to logarithmic phase, and then dispensed into 1ml culture tubes. Mu.l of phage positive clones were taken into the above 1ml culture tube. Shaking-culturing at 37 ℃ for 4.5-5 hours. The culture was transferred to a microcentrifuge tube and centrifuged for 30 seconds. The supernatant was transferred to a clean centrifuge tube, which was the amplified phage stock, and stored at 4 ℃ until the next experiment was not performed immediately.

And (3) rapid purification of a sequencing template: the phage-containing supernatant collected above (about 1ml in volume) was added with 400. mu.l of PEG/NaCl, mixed by inversion, left at room temperature for 10 minutes, centrifuged at 12,000rpm for 10 minutes, the supernatant was discarded, and centrifugation was carried out again, and the residual supernatant was thoroughly aspirated. The precipitate was added with 200. mu.l of iodide buffer (10mM Tris-HCl, 1mM EDTA, 4M NaI [ pH8.0]) and the precipitate was thoroughly resuspended, and then added with 500. mu.l of ethanol and incubated at room temperature for 10 minutes (to precipitate DNA); centrifuge at 12,000rpm for 10 minutes and discard the supernatant. Washing the precipitate with 1ml of pre-cooled 70% ethanol once, centrifuging at 12,000rpm for 10 min, removing the supernatant, opening the cover, and air drying overnight to fully volatilize ethanol. And finally, resuspending the precipitate in 30 mu l of double distilled water to obtain a sequencing template, detecting 5 mu l of the sequencing template by using 1% agarose gel electrophoresis, selecting-96 gIII sequencing primers from the DNA which is qualified by detection, sending the DNA to a company for sequencing, and analyzing the corresponding 12 peptide sequence of the phage.

By comparison, the applicant obtained a short peptide SEQ ID No. 1: YTNPYYSSHTRN are provided.

Example 4 cell ELISA for detecting binding of phage-displayed short peptides to cells

In order to verify the combination condition of the screened polypeptide and FR α expressed on the cell surface in a natural state, an ovarian cancer cell SKOV3 highly expressing FR α is selected, an irrelevant liver cancer cell HepG2 is set as a negative control, ELISA is carried out, the SKOV3 cell and the HepG2 cell are cultured until the cell density reaches more than 80%, the cell morphology is observed under a microscope to be good, the cell is digested by 0.25% of pancreatin, then corresponding fresh culture medium is added for basic suspension, viable cell counting is carried out, and the cell density is adjusted to 2 × 10⁵Each well was plated with 96-well cell culture plates at 100. mu.l per well, and incubated overnight at 37 ℃. The supernatant was aspirated off every other day and sterile PBS buffer (137mM NaCl, 2.7mM KCl, 10mM Na)₂HPO₄，2mM KH₂PO₄[PH7.4]) Washing twice with 5 min each, fixing with 10% paraformaldehyde at room temperature for 15 min, removing the fixing solution, and washing the plate with 5% of PBS buffer solution 5 min each for 3 timesSkim milk (PBS diluted) was used as a blocking solution and blocked at 37 ℃ for 2 hours. The blocking solution was discarded, the PBS was briefly rinsed, the corresponding phage was diluted appropriately and added to the cell culture plate, and a set of negative controls (wild type M13) was set up, 100. mu.l per well, and incubated overnight at 4 ℃. The supernatant was aspirated off every other day and PBST (TBS + volume ratio 0.1% [ v/v ]]Tween-20) and PBS buffer were washed 3 times for 5 minutes each. Then, 100. mu.l of HRP-labeled anti-M13 antibody diluted with blocking solution to a working concentration (1: 10000) was added to each well, and incubated at 37 ℃ for 2 hours, followed by washing as above, and then the chromogenic reaction and the termination reaction were carried out in the same phage ELISA procedure.

The results are shown in FIG. 3. Compared with wild M13, the experimental group polypeptide has better binding with SKOV3, and has obvious specificity (P < 0.05) compared with the binding with HepG2, which indicates that the screened FR alpha binding peptide can be specifically bound with the FR alpha expressed on the surface of tumor cells in a natural state.

Example 5 detection of binding of phage-displayed short peptides to cell surface FR α by flow cytometry

Purpose of the experiment cocellular ELISA experiments were performed. And detecting the binding condition of the screened positive phage monoclonal to SKOV3 tumor cells by using flow cytometry.

Cell treatment, culturing SKOV3 cells until cell density reaches above 80%, observing cell morphology under microscope, digesting with 0.25% pancreatin, adding fresh culture medium to stop digestion, centrifuging at 1,500rpm for 5 min, re-suspending with PBS to prepare single cell suspension, counting living cells, and adjusting cell concentration to 1-2 × 10⁶Each volume is divided into 1.5ml EP tubes, and each tube is 250-350 mul. And after centrifuging at 3,000rpm for 5 minutes, adding 100 mu l of diluted phage supernatant into each tube to serve as a primary antibody (the diluent is 2% FBS-PBS), selecting phage supernatant corresponding to the polypeptide obtained by sequencing, incubating for 1 hour at 4 ℃, taking out the phage supernatant at 3,000rpm, centrifuging for 5 minutes, discarding the supernatant, adding 1ml of PBS into each tube, washing for 2-3 times, and sucking and discarding the supernatant. Mu.l of diluted murine anti-M13 secondary antibody was added, left at 4 ℃ for 1 hour, removed and washed as above, and finally FITC-labeled goat anti-mouse fluorescent secondary antibody was added at a dilution ratio of 1: 500 (2% FBS-PBS) at 100. mu.l per well, taking care thatThis step requires the exclusion of light. Incubating at 4 ℃ in a dark place for 1 hour, washing the cells in the same way, adding a proper volume of PBS (phosphate buffer solution) for resuspension according to cell precipitation after the last washing, and detecting the cells by an up-flow cytometer.

From the binding results of flow cytometry, the phage supernatant was able to bind specifically to the cell line with high expression of FR α, as shown in fig. 4, the binding rate was 36%.

Claims

1. A polypeptide targeting folate receptor alpha, characterized in that the amino acid sequence is SEQ ID No. 1:

Tyr-Thr-Asn-Pro-Tyr-Tyr-Ser-Ser-His-Thr-Arg-Asn。

2. a nucleic acid encoding the polypeptide of claim 1.