CN107344964B - Affinity peptide P333 targeting AKAP3 - Google Patents

Abstract

The invention provides a polypeptide, the sequence of which is shown in SEQ ID NO. 1. The polypeptide of the invention is AKAP3 anti-tumor CTL epitope peptide, which can better induce specific CTL response and improve the expression quantity of IFN-gamma, thereby enhancing the effect of preventing and treating tumors and being prepared into a novel tumor therapeutic vaccine, even food and health care products.

Description

Affinity peptide P333 targeting AKAP3

Technical Field

The invention relates to the field of biology, in particular to a polypeptide.

Background

Type A Kinase Anchor Proteins (AKAPs) are a group of structurally distinct proteins that bind to the protein kinase a (pka) subunits. There is a large body of evidence that AKAPs play an important role in cAMP-mediated signaling. AKAP3 is a cancer testis antigen. Studies have shown that AKAP3 is a gene specific to sperm, expressed primarily after meiosis during spermatogenesis, and plays an important role in the morphological changes of sperm precursor cells and in the regulation of sperm function. In normal tissues, AKAP3 expression is only present in the testis. mRNA of AKAP3 is widely expressed in various tumor cell lines. It was found that AKAP3 is highly expressed in ovarian cancer and that expression of AKAP3 is associated with histological grade of the tumor and also with clinical stage of the tumor.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides an affinity peptide targeting AKAP3 or an anti-tumor CTL epitope peptide P333 derived from AKAP3 and application thereof.

In one aspect, the invention provides a polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 1.

In another aspect, the invention provides methods for preparing such polypeptides, which can be prepared by solid phase synthesis, e.g., using standard Fmoc protocols.

In another aspect, the invention provides a food, health product or pharmaceutical composition comprising the polypeptide, which may comprise the polypeptide and a pharmaceutically acceptable carrier or excipient thereof, which may be in the form of a vaccine.

In still another aspect, the invention provides the use of the polypeptide for preparing a prophylactic or therapeutic polypeptide vaccine for tumors positive for AKAP3 expression.

The invention has the beneficial effects that:

the polypeptide of the invention is AKAP3 anti-tumor CTL (cytotoxic T lymphocyte) epitope peptide, which can better induce specific CTL response and improve the expression quantity of IFN-gamma, thereby enhancing the effect of tumor prevention and treatment and being prepared into a novel tumor therapeutic vaccine, even food and health care products.

Drawings

FIG. 1 shows that ELISPOT method detects the capability of epitope peptide in vitro specific CTL to secrete IFN-gamma;

FIG. 2 depicts LDH assay for epitope peptide in vitro specific CTL cytotoxicity;

FIG. 3 depicts the ELISPOT method for detecting the IFN-gamma secretion ability of epitope peptide in vivo specific CTL;

FIG. 4 depicts LDH assay for epitope peptide in vivo specific CTL cytotoxicity;

FIG. 5 shows the measurement of IFN-. gamma.secretion in mouse serum by ELISA;

FIG. 6 is a graph of the change in body weight of transgenic mice.

Reference P in each figure<0.05，**P<0.01，***P<0.001 represents the significant difference between the experimental group and the PBS group; a-up of P<0.05，▲▲P<0.01 and a-solidup-P<0.001 represents the experimental group and HBcAg, respectively_18–27Or significant differences in the group of Th table bits.

Detailed Description

The sequence of the candidate peptide P333 is LIDSFLRNL (Leu-Ile-Asp-Ser-Phe-Leu-Arg-Asn-Leu), as shown in SEQ ID NO.1, the candidate peptide P333 is synthesized by a standard Fmoc scheme in a solid phase, and the molecular weight is detected by mass spectrum: 1091.7[ M + H ], basically the same as theoretical value 1090.3. The activity of the candidate peptide (sometimes also referred to as epitope peptide) is identified below, using experimental methods and reagents that are routinely selected, see literature: shi RR, Liu J, Zou Z, Qi YM, ZHai MX, ZHai WJ, GaoYF The immunogenicity of a novel cytoxic T lymphocyte epitope from a genomic DNA PL2L60 complex benzene enhanced by 4-chlorophenylalanine inhibition position 1.Cancer immunization, immunotherapy: CII 2013,62(11): 1723-. Unless otherwise defined, all terms and abbreviations used herein have the same meaning as commonly understood in the art.

T2A2 cell affinity assay

Collecting T2A2 cells, washed 3 times with serum-free IMDM, adjusted cell concentration to 1X 10⁶Per mL, plated in 24-well plates, 1 mL/well. The experimental group was added with 2.5. mu.g/mL of beta₂Microglobulin, 50 μ M candidate peptide P333; setting a positive group, a negative group and a background control group. At 37 ℃ with 5% CO₂And co-incubating for 18h in the incubator.

② collecting the incubated cells, washing with ice PBA for 3 times, adding 0.5mL of monoclonal antibody BB7.2 diluted by 1:100, incubating for 30min at 4 ℃ in the dark.

③ washing with cold PBA for 3 times, adding 50 μ L of a 1:50 dilution of FITC-goat anti-mouse IgG solution, and incubating at 4 ℃ in the dark for 40 min. The cold PBA was washed 1 time, 1mL of PBA was resuspended, and examined on the flow cytometer. At the same time, the identified HBcAg is set_18-27As a positive peptide for the binding capacity and stability experiments, PBS was used as a negative control, and pure T2a2 cells without peptide stimulation were used as a background control.

The results are expressed in Fluorescence (FI): fluorescence coefficient (FI) ═ average (epitope peptide average fluorescence intensity-background average fluorescence intensity)/background average fluorescence intensity. And (4) analyzing results: if FI > 1.5: candidate peptides have strong affinity to HLA-a 0201; 1.5> FI > 0.5: moderate affinity; FI < 0.5: weak affinity.

The peptide was analyzed to have FI of 2.86.

2. peptide/MHC complex stability assay

T2A2 cells were collected, washed 3 times with serum-free IMDM, and cell concentration was adjusted to 1X 10⁶Per mL, plated in 24-well plates, 1 mL/well. Adding 50 μ M candidate peptide, setting positive group, negative group, background group and zero-adjusting group, adding 2.5 μ g/mL beta₂Microglobulin, 5% CO at 37 ℃₂And co-incubating for 18h in the incubator.

After the incubation was complete, the unbound peptides were washed 3 times with cold PBA and incubated for 1h with the addition of 10. mu.g/mL brefeldin A. Taking 0h, 2h, 4h and 6h as time points, at 37 deg.C and 5% CO²And (4) co-incubation. The PBA was washed 2 times with cold PBA, and 500. mu.L of monoclonal antibody BB7.2 diluted at 1:100 was added, and incubated at 4 ℃ for 30min in the dark. Washing with cold PBA for 2 times, adding 50 μ L of FITC-goat anti-mouse IgG at 1:50 dilution, incubating at 4 deg.C in dark for 40min. After cold PBA washes, PBA resuspended cells, examined in a flow cytometer, and the results were expressed as DCs₅₀And (4) showing.

Upon detection, DC₅₀>6h。

3. In vitro immune activity assay

In vitro induction of cytotoxic T lymphocytes

Adding a proper amount of heparin sodium solution into a 40mL centrifuge tube, extracting 20mL of peripheral blood of an HLA-A2 positive healthy volunteer, and diluting the anticoagulated blood by using PBS (sterile pH 7.2).

② 4mL of lymphocyte splitting liquid is slowly added into a sterile centrifuge tube along with the anticoagulation diluted by PBS, and the interface is not broken. Centrifuge at 2000rpm for 20 min.

After the centrifugation is finished, the cells in the centrifugal tube are divided into four layers from top to bottom; the first plasma layer was discarded and the second annular opalescent lymphocyte layer was aspirated into another sterile centrifuge tube and washed with 5 volumes of PBS at 2000rpm for 15 min.

And fourthly, abandoning the supernatant and collecting the precipitate. Cell concentration was adjusted to 1X 10 using IMDM medium containing 10% fetal bovine serum⁶Perml, cultured in 24-well plates.

Fifthly, setting the PBS group, the HBV group and the experimental group, and respectively adding corresponding polypeptide and beta to the HBV group and the experimental group on the 2 nd day₂And (4) M (setting the final concentration of both to be 10 mu g/mL), adding corresponding PBS into the PBS group, adding 50U/mL recombinant human source IL-2(rh IL-2) on the 3 rd day, observing the state of the culture solution, carrying out half-amount solution change, and supplementing the rh IL-2. One round of repeated stimulation is carried out every 7 days, and the stimulation process comprises the following steps: the 24-well plate was centrifuged at 1000rpm for 10min to remove the supernatant, fresh IMDM medium containing 10% fetal calf serum was added, while the amounts of the corresponding polypeptide, rh IL-2 and β 2-M were supplemented, and CD3 antibody was added on day 3 of the 2 nd round stimulation.

Sixthly, after finishing 3 times of stimulation, continuously culturing for 2-3 days, and collecting cells to be used as the effect CTL.

Detection of immunological Activity

1) ELISPOT (Enzyme-linked Immunospot Assay) method for detecting the number of IFN-gamma-secreting T cells

a. First, activation of the pre-coated plate is performed:

taking out the required ELISPOT lath, adding 200 μ L of serum-free IMDM culture medium into each hole for sealing, standing for 10min, and discarding the culture medium;

b. cell plating:

induced CTL as effector cells and peptide-bearing T2A2 as stimulator cells, all at 2X 10 cell concentrations⁶Per mL; each well of the experimental hole is added with 50 mu L of effector cells and 50 mu L of stimulating cells;

adding 100 mu L of serum-free IMDM culture medium to each well of the negative control wells;

positive control wells were plated with 50. mu.L of effector cells and 10. mu.L PHA supplemented with 40. mu.L of serum-free IMDM medium.

Placing in incubator at 37 deg.C and 5% CO₂Incubation for 18 h;

c. cell lysis:

after the incubation is finished, pouring out the culture medium in the holes, adding 200 mu L of sterile deionized water into each hole, and cracking the cells for 10min at 4 ℃; pouring out the liquid in the holes, adding 200 mu L of 1 × Washing Buffer for Washing, Washing for 6 times, staying for 60s each time, and after Washing, patting the water-absorbent paper dry;

d. adding a detection antibody for incubation:

adding 100 mu L of biotin-labeled antibody, and incubating for 1h at 37 ℃; after incubation, pouring out liquid in the holes, adding 200 mu L of 1 × Washing Buffer for Washing, Washing for 6 times, staying for 60s each time, and after Washing, patting dry on absorbent paper;

e. adding enzyme-linked avidin for incubation:

adding 100 mu L of enzyme-linked avidin, and incubating for 1h at 37 ℃; after incubation, pouring out liquid in the holes, adding 200 mu L of 1 multiplied by Washing Buffer into each hole for Washing, Washing for 6 times, staying for 60s each time, and drying on absorbent paper after Washing;

f. color development:

adding 100 μ L of AEC color developing solution, standing at 25 deg.C in dark for 30min, and developing; after the color development is finished, pouring out the liquid in the hole, drilling a hole plate base, washing with deionized water, and stopping the color development. Placing the mixture in a ventilated place, standing and drying at room temperature; the number of spots per well in a 96-well plate was counted using an ELISPOT image analyzer and the results are shown in fig. 1 for comparison.

2) Experiment for detecting cytotoxicity by LDH (Lactate Dehydrogenase) method

Preparation of target cells and Effector cells

In this experiment, SW620 was used as a target cell, and the cell concentration was adjusted to 1X 10⁵Per mL, 5000/50 μ L per well, different numbers and volumes of effector cells (prepared by in vitro induction) were added to form different effective-to-target ratios in the experimental groups (12.5: 1, 25:1, 50:1, respectively), and serum-free IMDM medium was added to a total volume of 100 μ L, with 3 duplicate wells per group.

The following controls were also set up:

target cell spontaneous release group

Maximum release group of target cells

Effector cell spontaneous release group

Background control group

Total volume correction control groups were also provided with 3 duplicate wells, each with a total volume of 100. mu.L per well.

LDH experiment step:

the 96-well plate of the cell culture plate is placed at 37 ℃ and 5% CO₂Culturing in an incubator for 4 h. 45min before the end of incubation, 10. mu.L of 10 × lysate was added to the target cell maximum release well and the volume corrected well. After the culture is finished, centrifuging the culture plate at 1000rpm for 10 min; taking out the 96-well plate, slightly sucking 50 mu L of supernatant from each well, placing the supernatant into a corresponding well of another 96-well plate, quickly adding 50 mu L of lactate dehydrogenase substrate mixed liquor into each well after all the transfer is finished, and incubating for 30min at room temperature in a dark place; after incubation, 50 μ L of stop solution was added to each well; the absorbance OD was measured at 490nm on a microplate reader to calculate the killing rate.

The killing rate is (experimental group release-effector cell spontaneous release-target cell spontaneous release)/(target cell maximal release-target cell spontaneous release) × 100%.

The results are shown in FIG. 2.

HLA-A2.1/K of AKAP3^bDetection of immune Activity in transgenic mice

In vivo Induction of effector CTL

Randomly selecting HLA-A2.1/K of 6-8 weeks old^bTransgenic mice, 5 per group, were randomly assigned males and females.

The PBS group, the Th epitope group and the experimental group are set in the experiment. The doses in each group of injected mice were as follows:

PBS group: PBS: freund's incomplete adjuvant (IFA) ═ 1:1

Group Th: PBS: th epitope: freund's incomplete adjuvant (IFA) ═ 1:1:2

Experimental groups: experimental groups: th epitope: freund's incomplete adjuvant (IFA) ═ 1:1:2

Day 0 for the 1 st immunization, subcutaneous immunizations were given as above for each fraction, and 2 nd and 3 rd boosts were given at 5d and 10 d; at the same time, before each immunization injection, the antigen is applied to HLA-A2.1/K^bTransgenic mice were observed and body weights were recorded and the results are shown in figure 6.

Preparing effector cells:

firstly, HLA-A2.1/K after immune injection^bKilling the transgenic mouse after removing neck at 11d, soaking in 75% alcohol for 10min for sterilization, taking out spleen after aseptic abdominal cavity opening, removing fat and connective tissue, and placing in a 200-mesh stainless steel mesh screen;

② adding a small amount of PBS with pH 7.2 into the culture dish, placing 200 meshes of steel mesh on the culture dish, grinding by using a 20mL medical syringe inner core, and grinding to the utmost extent. Washing with PBS, removing the screen, obtaining spleen cell suspension (about 10mL) in a culture dish, and transferring the spleen cell suspension into a sterile centrifuge tube;

③ horizontally centrifuging at 1000rpm for 10min, abandoning the supernatant and collecting the cells; adding 5mL of erythrocyte lysate into each tube, gently blowing and beating the resuspended cells, and incubating for 15min at 4 ℃ to lyse the erythrocytes; the supernatant was centrifuged at 1000rpm for 10min and the cells were collected. PBS wash 2 times;

fourthly, the spleen cells are suspended in 10mL RPMI 1640 medium containing 10% fetal bovine serum. Culturing the counted spleen cell suspension in a 6-pore plate, wherein each pore is 5mL of cell suspension; adding 250U recombinant murine IL-2, beta per well the next day₂-M and the corresponding polypeptide; at 37 deg.C, 5% CO₂Culturing in an incubator for 5d, and taking the harvested cells as effector cells to carry out ELISPOT and LDH detection. The ELISPOT and LDH detection methods are the same as those used in the in vitro immunization experiment, and the detection results of the ELISPOT method are shown in figure 3, and the detection results of the LDH method are shown in figure 4.

HLA-A2.1/K^bIFN-gamma enzyme-linked immunoassay (ELISA) in serum of transgenic mice

Before transgenic mice are killed after cervical dislocation, venous blood of each group of immune mice is taken by an eyeball blood taking method, after standing for 2-4h at room temperature, centrifuging for 10min at 3000rpm, taking upper serum, and storing at-80 ℃ after marking. The ELISA method was used to detect IFN-. gamma.antibody levels in mouse serum, and the results are shown in FIG. 5.

Sequence listing

<110> higher specialty school of Loxowo medicine

<120> affinity peptide P333 targeting AKAP3

<160>1

<210>1

<211>9

<212>PRT

<213> Artificial Synthesis

<400>1

Leu Ile Asp Ser Phe Leu Arg Asn Leu

1 5

Claims (1)

1. The amino acid sequence of the polypeptide is shown in SEQ ID NO. 1.

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